Methods of analyzing a biological sample

ABSTRACT

Chemically reactive carbocyanine dyes that are intramolecularly crosslinked between the 1-position and 3′-position, their bioconjugates and their uses are described. 1,3′-crosslinked carbocyanines are superior to those of conjugates of spectrally similar 1,1′-crosslinked or non-crosslinked dyes. The invention includes derivative compounds having one or more benzo nitrogens.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. patent application Ser. No.11/256,581, filed Oct. 21, 2005, and to its priority document, U.S.Provisional Patent Application Ser. No. 60/621,789, filed Oct. 25, 2004,the entire disclosures of which are incorporated by reference.

FIELD OF THE INVENTION

The invention relates to fluorescent chemicals, including reactive dyesand dye-conjugates; and to their uses.

BACKGROUND OF THE INVENTION

Luminescent probes are valuable reagents for the analysis and separationof molecules and cells and for the detection and quantification of othermaterials. A very small number of luminescent molecules can be detectedunder optimal circumstances. Barak and Webb visualized fewer than 50fluorescent lipid analogs associated with the LDL reception of cellsusing a SIT camera, J. CELL BIOL., 90, 595-604 (1981). Flow cytometrycan be used to detect fewer than 10,000 fluorescein molecules associatedwith particles or certain cells (Muirhead, Horan and Poste,BIOTECHNOLOGY, 3, 337-356 (1985)). Some specific examples of theapplication of fluorescent probes are (1) identification and separationof subpopulations of cells in a mixture of cells by the techniques offluorescence flow cytometry, fluorescence-activated cell sorting andfluorescence microscopy; (2) determination of the concentration of asubstance that binds to a second species (e.g., antigen-antibodyreactions) in the technique of fluorescence immunoassay; (3)localization of substances in gels and other insoluble supports by thetechniques of fluorescence staining. These techniques are described byHerzenberg, et al., “CELLULAR IMMUNOLOGY” 3rd ed., Chapter 22; BlackwellScientific Publications (1978); and by Goldman, “FLUORESCENCE ANTIBODYMETHODS”, Academic Press, New York, (1968); and by Taylor, et al.,APPLICATIONS OF FLUORESCENCE IN THE BIOMEDICAL SCIENCES, Alan Liss Inc.,(1986).

When employing fluorescent dyes for the above purposes, there are manyconstraints on the choice of the fluorescent dye. One constraint is theabsorption and emission characteristics of the fluorescent dye, sincemany ligands, receptors, and materials in the sample under test, e.g.blood, urine, cerebrospinal fluid, will fluoresce and interfere with anaccurate determination of the fluorescence of the fluorescent label.This phenomenon is called autofluorescence or background fluorescence.Another consideration is the ability to conjugate the fluorescent dye toligands and receptors and other biological and non-biological materialsand the effect of such conjugation on the fluorescent dye. In manysituations, conjugation to another molecule may result in a substantialchange in the fluorescent characteristics of the fluorescent dye and, insome cases, substantially destroy or reduce the quantum efficiency ofthe fluorescent dye. It is also possible that conjugation with thefluorescent dye will inactivate the function of the molecule that islabeled. A third consideration is the quantum efficiency of thefluorescent dyes which should be high for sensitive detection. A fourthconsideration is the light absorbing capability, or extinctioncoefficient, of the fluorescent dyes, which should also be as large aspossible. Also of concern is whether the fluorescent molecules willinteract with each other when in close proximity, resulting inself-quenching. An additional concern is whether there is non-specificbinding of the fluorescent dyes to other compounds or container walls,either by themselves or in conjunction with the compound to which thefluorescent dye is conjugated.

The applicability and value of the methods indicated above are closelytied to the availability of suitable fluorescent compounds. Inparticular, there is a need for fluorescent substances that emit in thelonger wavelength region (yellow to near infrared), since excitation ofthese chromophores produces less autofluorescence and also multiplechromophores fluorescing at different wavelengths can be analyzedsimultaneously if the full visible and near infrared regions of thespectrum can be utilized. Fluorescein, a widely used fluorescentcompound, is a useful emitter in the green region although in certainimmunoassays and cell analysis systems background autofluorescencegenerated by excitation at fluorescein absorption wavelengths limits thedetection sensitivity. However, the conventional red fluorescent labelrhodamine has proved to be less effective than fluorescein.

Phycobiliproteins have made an important contribution because of theirhigh extinction coefficient and high quantum yield. Thesechromophore-containing proteins can be covalently linked to manyproteins and are used in fluorescence antibody assays in microscopy andflow cytometry. The phycobiliproteins have the disadvantages that (1)the protein labeling procedure is relatively complex; (2) the proteinlabeling efficiency is not usually high (typically an average of 0.5phycobiliprotein molecules per protein); (3) the phycobiliproteins arenatural products and their preparation and purification are complex; (4)the phycobiliproteins are expensive; (5) there are at present nophycobiliproteins available as labeling reagents that fluoresce furtherto the red region of the spectrum than allophycocyanine, whichfluoresces maximally at 680 nm; (6) the phycobiliproteins are largeproteins with molecular weights ranging from 33,000 to 240,000 and arelarger than many materials that are desirable to label, such asmetabolites, drugs, hormones, derivatized nucleotides, and many proteinsincluding antibodies. The latter disadvantage is of particularimportance because antibodies, avidin, DNA-hybridization probes,hormones, and small molecules labeled with the large phycobiliproteinsmay not be able to bind to their targets because of steric limitationsimposed by the size of the conjugated complex.

Other techniques involving histology, cytology, immunoassays would alsoenjoy substantial benefits from the use of a fluorescent dye with a highquantum efficiency, absorption and emission characteristics at longerwavelengths, having simple means for conjugation and being substantiallyfree of nonspecific interference.

Fluorescent compounds are covalently or noncovalently attached to othermaterials to impart color and fluorescence. Brightly fluorescent dyespermit detection or location of the attached materials with greatsensitivity. Certain carbocyanine dyes have demonstrated utility aslabeling reagents for a variety of biological applications, e.g. U.S.Pat. No. 4,981,977 to Southwick, et al. (1991); U.S. Pat. No. 5,268,486to Waggoner, et al. (1993); U.S. Pat. No. 5,569,587 to Waggoner (1996);U.S. Pat. No. 5,569,766 to Waggoner, et al. (1996); U.S. Pat. No.5,486,616 to Waggoner, et al. (1996); U.S. Pat. No. 5,627,027 toWaggoner (1997); U.S. Pat. No. 5,808,044 to Brush, et al. (1998); U.S.Pat. No. 5,877,310 to Reddington, et al. (1999); U.S. Pat. No. 6,002,003to Shen, et al. (1999); U.S. Pat. No. 6,004,536 to Leung, et al. (1999);U.S. Pat. No. 6,008,373 to Waggoner, et al. (1999); U.S. Pat. No.6,043,025 to Minden, et al. (2000); U.S. Pat. No. 6,127,134 to Minden,et al. (2000); U.S. Pat. No. 6,130,094 to Waggoner, et al. (2000); U.S.Pat. No. 6,133,445 to Waggoner, et al. (2000); also WO 97/40104, WO99/51702, WO 01/21624, and EP 1 065 250 A1; and TETRAHEDRON LETT., 41,9185-88 (2000). Nevertheless, many carbocyanine dyes are known to sharecertain disadvantages, e.g. severe quenching of the fluorescence ofcarbocyanine dyes in biopolymer conjugates, e.g. quenching of Cy5 andCy7 dye variants on conjugates, as discussed by Gruber, et al.,BIOCONJUGATE CHEM., 11, 696 (2000), and in EP 1 065 250 A1, 0004. Inaddition, certain desired sulfoalkyl derivatives of the reactivecarbocyanine dyes are difficult to prepare, as indicated for Cy3 and Cy5variants by Waggoner and colleagues in BIOCONJUGATE CHEM., 4, 105, 109(1993). Cyanine dyes also have a very strong tendency to self-aggregate(i.e. stack), which can significantly reduce the fluorescence quantumyields, as described in the extensive review by Mishra, et al., CHEM.REV., 100, 1973 (2000).

Another problem with the existing carbocyanine labeling dyes is the freerotation/vibration of two indolium (or benzothiazolium, orbenzoimidazolium) heads around the middle conjugated double bonds thatsignificantly reduce their fluorescence intensities (see Scheme 1). Thisphenomenon is called ‘loose belt effect’ that is described in “MODERNMOLECULAR PHOTOCHEMISTRY”, Chapters 5 and 6, University Science Books,Sausalito, Calif., authored by Nicholas J. Turro (1991).

This so-called ‘loose belt effect’ can be eliminated by the crosslinkingof the two heads. 1,1′-crosslinking of cyanines is disclosed by R.Singh, et al. WO 01/02374 (2001), which is supposed to eliminate the‘loose belt effect’ described above. However, we observe that the1,1′-crosslinking actually causes the decreased fluorescence quantumyield of dye-protein conjugates compared to that of non-crosslinkedcarbocycanine-protein conjugates at the similar ratios of dye/protein(see FIG. 3). This unfavorable fluorescence quantum decrease might becaused by the inappropriate stereochemistry of 1,1′-crosslinking.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Absorption spectra of Cy5 free acid (from Amersham Biosciences)and Compound 13 in PBS buffer (pH=7.4). Absorption characteristics ofthe Compound 13 are similar to those of Cy5, when present as thefree-acid.

FIG. 2. Fluorescence spectra of Cy5 free acid (from AmershamBiosciences) and Compound 13 in PBS buffer (pH=7.4, excited at 630 nm).Fluorescence characteristics of the Compound 13 are similar to those ofCy5, when present as the free-acid.

FIG. 3. Comparison of fluorescence quantum yields of Cy5 SE and Compound38 when conjugated to goat anti-rabbit IgG (GAR). The conjugates areprepared and characterized as described in Examples 58.

FIG. 4. Comparison of fluorescence quantum yields of Cy5 SE andCompounds 14 and 38 when conjugated to goat anti-rabbit IgG (GAR). Theconjugates are prepared and characterized as described in Examples 58.

FIG. 5. Photostability comparison of Compound 13 (solid circles) withCy5 free acid (squares) in PBS buffer (pH 7.4). The detailedexperimental conditions are described in Example 64.

FIG. 6. Synthesis of a cyanine that has a RGM at 1-position.

FIG. 7. Synthesis of a cyanine that has a RGM at 3′-position.

FIG. 8. Synthesis of a cyanine that has a RGM at the non-conjugatedChain C.

FIG. 9. Synthesis of a cyanine that has a RGM at the conjugated doublebond bridge.

FIG. 10. Synthesis of a cyanine that has a RGM at Ring A or Ring B

FIG. 11. Synthesis of a cyanine through intramolecular coupling.

SUMMARY OF THE INVENTION AND DESCRIPTION OF PREFERRED EMBODIMENTS

We discovered that 1,3′-crosslinking of an carbocyanine dye unexpectedlymitigates problems discussed in the background section and results indye-polymer conjugates that are substantially more fluorescent onproteins, nucleic acids and other biopolymers, than conjugates labeledwith structurally similar 1,1′-crosslinked carbocyanine ornon-crosslinked dyes (see FIG. 4). The enhanced fluorescence intensityof dye-biomolecule conjugates of the invention results in greater assaysensitivity. The increase in fluorescence quantum yields may result fromthe reduction of the ground state aggregation caused by thesterohindrance of 1,3′-crosslinking of an carbocyanine dye. Thisintramolecular 1,3′-crosslinking might also reduce the oxidativedimerization of carbocyanines, and thus decrease their sensitivity toozone (see T. Katoh, et al. BULL. CHEM. SOC. JPN., 70, 1109-1114(1997)). The increased ozone resistance provides a great advantage fortheir applications of the claimed dyes in microarrays. The ozonesensitivity of carbocyanines has been a serious problem for themicroarray applications of Cy3, Cy5 and their analogs.

In addition to having more intense fluorescence emission thanstructurally similar cyanine dyes at similar wavelengths, and decreasedartifacts in their absorption spectra upon conjugation to biopolymers,certain embodiments of the invention also have greater photostability(see FIG. 5) and higher absorbance (extinction coefficients) at thewavelength(s) of peak absorbance than such structurally similar dyes.The enhanced photostability might also be related to the reduction ofoxidative dimerization. These improvements result in significantlygreater sensitivity in assays that use these dyes and their conjugates,while utilizing existing filters and instrumentation alreadycommercially available for use with similar dyes such as Cy3, Cy5, Cy5.5and Cy7.

Furthermore, the dyes of the invention typically exhibit absorbancemaxima between about 530 nm and about 800 mm, so these dyes can beselected to match the principal emission lines of the mercury arc lamp(546 nm), frequency-doubled Nd-Yag laser (532 nm), Kr-ion laser (568 nmand 647 nm), HeNe laser (543 mm, 594 nm, and 633 nm) or long-wavelengthlaser diodes (especially 635 nm and longer). Some dyes of the inventionexhibit very long wavelength excitation (at least 640 mm, but somegreater than about 730 nm) and emission bands (at least 665 nm, and somegreater than about 750 mm), so they are particularly useful for samplesthat are transparent to infrared wavelengths.

The present invention comprises reactive 1,3′-crosslinked carbocyaninedyes and their conjugates. The dyes and dye conjugates are used tolocate or detect the interaction or presence of analytes or ligands in asample. Kits incorporating such dyes or dye conjugates facilitate theiruse in such methods.

The dyes of the invention typically have Formula I:

wherein rings A and B represent the atoms necessary to form anitrogen-containing five-membered heterocyclic ring that has zero tothree fused aromatic rings; and each said fused aromatic ring selectedfrom the group consisting of C, CH, C(alkyl), O, S, N(aryl) andN(alkyl), and said five-membered ring contains ═N(alkyl) coupled to thebridged and conjugated double bonds, and said aromatic rings areoptionally substituted one or more times by substituents selected fromthe group consisting of a hydrogen, an alkyl having 1-20 carbons, ahydroxy, an alkoxy having 1-20 carbons, a trifluoromethyl, a halogen, amethylthio, a sulfonyl, a carbonyl, a hydroxy, an amino, a thiol, asulfate, a phosphonate or a RGM

C is a non-conjugated chain of 10-50 linear atoms selected from carbon,nitrogen, oxygen, phosphorus and sulfur that are further substituted bya hydrogen, an alkyl having 1-20 carbons, a hydroxy, an alkoxy having1-20 carbons, a trifluoromethyl, a halogen, a methylthio, a sulfonyl, acarbonyl, a hydroxy, an amino, an alkylamino, an arylamino, a thiol, asulfate, a phosphonate or a RGM.

n is 0 to 3.

R₁, R₂, R₃ and R₄ are independently selected from the group consistingof a hydrogen, an alkyl having 1-20 carbons, a cycloalkyl having 3-20carbons, an aryl, a heteroaryl, an amino, an alkylamino, an arylamino, athiol and a RGM.

RGM is a chemically reactive group described below.

The dyes of the invention comprise a cyanine dye that contains: 1) a RGMgroup; and 2) a bridged and non-conjugated chain C that intramolecularlycrosslinks position 1 (ring A) with position 3′ (ring B). In oneembodiment of the invention, the first or second ring system issubstituted by a side chain at position 1 that contains a RGM group. Inanother embodiment, the first or second ring contains a RGM groupdirectly located on the aromatic rings (A or B). In another embodiment,the bridged methine is substituted by a side chain that contains a RGMgroup. In another embodiment, the bridged and non-conjugated chain C issubstituted by a side chain that contains a RGM group. In anotherembodiment, the carbon atom at position 3 or 3′ is substituted by a sidechain that contains a RGM group.

Preferred compounds have at least one substituted indolium ring systemwherein the substituent contains a RGM and a non-conjugated bridgedchain. Other preferred compounds incorporate at least a charged group(e.g., sulfonate and ammonium moieties) to increase water solubility. By“sulfo” is meant sulfonic acid, or salt of sulfonic acid (sulfonate).Similarly, by “carboxy” is meant carboxylic acid or salt of carboxylicacid. “phosphate”, as used herein, is an ester of phosphoric acid, andincludes salts of phosphate. “phosphonate”, as used herein, meansphosphonic acid and includes salts of phosphonate. As used herein,unless otherwise specified, the alkyl portions of substituents such asalkyl, alkoxy, arylalkyl, alylamino, dialkylamino, trialkylammonium, orperfluoroalkyl are optionally saturated, unsaturated, linear orbranched, and all alkyl, alkoxy, alkylamino, and dialkylaminosubstituents are themselves optionally further substituted by carboxy,sulfo, amino, or hydroxy.

A preferred embodiment is a compound of Formula II:

wherein C is a non-conjugated chain of 10-50 linear atoms selected fromcarbon, nitrogen, oxygen; phosphorus and sulfur that are furthersubstituted by a hydrogen, an alkyl having 1-20 carbons, an alkoxyhaving 1-20 carbons, a trifluoromethyl, a halogen, a methylthio, asulfonyl, a carbonyl, a hydroxy, an amino, a thiol or a RGM. R₁ to R₁₆are a hydrogen, an alkyl having 1-20 carbons, an alkoxy having 1-20carbons, a trifluoromethyl, a halogen, a methylthio, a sulfonyl, acarbonyl, a hydroxy, an amino, a thiol or a RGM; X is O, S, Se, NR₁₅ orCR₁₅R₁₆; n is 0 to 3.

Another preferred embodiment is a compound of Formula III:

wherein C is a non-conjugated chain of 10-50 linear atoms selected fromcarbon, nitrogen, oxygen, phosphorus and sulfur that are furthersubstituted by a hydrogen, an alkyl having 1-20 carbons, an alkoxyhaving 1-20 carbons, a trifluoromethyl, a halogen, a methylthio, asulfonyl, a carbonyl, a hydroxy, an amino, a thiol or a RGM. R₁ to R₁₆are a hydrogen, an alkyl having 1-20 carbons, an alkoxy having 1-20carbons, a trifluoromethyl, a halogen, a methylthio, a sulfonyl, acarbonyl, a hydroxy, an amino, a thiol or a RGM; X is O, S, Se, NR₁₅,CR₁₅R₁₆; n is 0 to 3.

Another preferred embodiment is a compound of Formula IV:

wherein C is a non-conjugated chain of 10-50 linear atoms selected fromcarbon, nitrogen, oxygen, phosphorus and sulfur that are furthersubstituted by a hydrogen, an alkyl having 1-20 carbons, an alkoxyhaving 1-20 carbons, a trifluoromethyl, a halogen, a methylthio, asulfonyl, a carbonyl, a hydroxy, an amino, a thiol or a RGM. R₁ to R₁₆are a hydrogen, an alkyl having 1-20 carbons, an alkoxy having 1-20carbons, a trifluoromethyl, a halogen, a methylthio, a sulfonyl, acarbonyl, a hydroxy, an amino, a thiol or a RGM; X is O, S, Se, NR₁₅,CR₁₅R₁₆; n is 0 to 3.

Another preferred embodiment is a compound of Formula V:

wherein C is a non-conjugated chain of 10-50 linear atoms selected fromcarbon, nitrogen, oxygen, phosphorus and sulfur that are furthersubstituted by a hydrogen, an alkyl having 1-20 carbons, an alkoxyhaving 1-20 carbons, a trifluoromethyl, a halogen, a methylthio, asulfonyl, a carbonyl, a hydroxy, an amino, a thiol or a RGM. R₁ to R₁₆are a hydrogen, an alkyl having 1-20 carbons, an alkoxy having 1-20carbons, a trifluoromethyl, a halogen, a methylthio, a sulfonyl, acarbonyl, a hydroxy, an amino, a thiol or a RGM; X is O, S, Se, NR₁₅,CR₁₅R₁₆; n is 0 to 3.

The length of the conjugated polymethine bridge between the two ringsystems greatly affects the dye's absorption and emission properties.Each of R₁, R₂, R₃, when present, is independently a hydrogen, a fluoro,a chloro, an alkyl having 1-6 carbons, an alkoxy having 1-6 carbons, anaryloxy, a N-heteroaromatic moiety, or an iminium ion. Alternatively,two substituents R₁/R₂, R₂/R₃, when taken in combination, form a 4-, 5-,or 6-membered saturated or unsaturated hydrocarbon ring that isunsubstituted or is optionally substituted one or more times by asaturated or unsaturated alkyl having 1-6 carbons, a halogen, or acarbonyl oxygen. Typically, each of R₁, R₂ and R₃, when present, is ahydrogen. Where one of R₁, R₂ and R₃ is a nonhydrogen, it is typicallythe substituent on the center carbon of bridged and conjugated doublebonds. Similarly, where bridged and conjugated double bonds incorporatea 4-, 5-, or 6-membered ring, it typically occurs at the center of theconjugated bridge moiety.

Additionally, selection of the A, B and X moieties may alsosignificantly affect the dye's absorption and fluorescence emissionproperties. A and B optionally the same or different, and spectralproperties of the resulting dye may be tuned by careful selection of Aand B. In one embodiment, X is CR₁₅R₁₆ where R₁₅ and R₁₆ are a hydrogenor an alkyl group having 1-30 carbons, that is optionally substitutedone or more times by a hydroxy, a carboxy, a sulfo, an amino, analkylamino having 1-6 carbons or dialkylamino having 2-20 carbons.Alternatively, R₁₅ and R₁₆ in combination complete a five or sixmembered saturated or unsaturated ring that is optionally substituted bya RGM. Preferably R₁₅ and R₁₆ are independently an alkyl with 1-6 carbonatoms that are unsubstituted or are substituted once by a hydroxy, asulfo, a carboxy or an amino. In one aspect of the invention, R₁₅ andR₁₆ are alkyls having 1-6 carbons, preferably methyls. In another aspectof the invention, one of R₁₅ and R₁₆ is a methyl, and the other is analkyl having 1-10 carbons that is substituted by a carboxy or by a sulfoor by a hydroxy, or by a RGM.

Incorporation of one or more non-hydrogen substituents on the fusedrings can be used to fine tune the absorption and emission spectrum ofthe resulting dye.

Another preferred embodiment of the invention is a compound of FormulaVI

wherein C is a non-conjugated chain of 10-50 linear atoms selected fromcarbon, nitrogen, oxygen, phosphorus and sulfur that are furthersubstituted by a hydrogen, an alkyl having 1-20 carbons, an alkoxyhaving 1-20 carbons, a trifluoromethyl, a halogen, a methylthio, asulfonyl, a carbonyl, a hydroxy, an amino, a thiol or a RGM. R₁ to R₂₇are a hydrogen, an alkyl having 1-20 carbons, an alkoxy having 1-20carbons, a trifluoromethyl, a halogen, a methylthio, a sulfonyl, acarbonyl, a hydroxy, an amino, a thiol or a RGM; X is O, S, Se, NR₁₅ orCR₁₅R₁₆; n is 0 to 3.

Another preferred embodiment of the invention is a compound of FormulaVII

wherein C is a non-conjugated chain of 10-50 linear atoms selected fromcarbon, nitrogen, oxygen, phosphorus and sulfur that are furthersubstituted by a hydrogen, an alkyl having 1-20 carbons, an alkoxyhaving 1-20 carbons, a trifluoromethyl, a halogen, a methylthio, asulfonyl, a carbonyl, a hydroxy, an amino, a thiol or a RGM. R₁ to R₂₃are a hydrogen, an alkyl having 1-20 carbons, an alkoxy having 1-20carbons, a trifluoromethyl, a halogen, a methylthio, a sulfonyl, acarbonyl, a hydroxy, an amino, a thiol or a RGM; X is O, S, Se, NR₁₅ orCR₁₅R₁₆; n is 0 to 3.

In one aspect of the invention, one or two or more of R₁ to R₂₇ is anamino, a carboxy and a thiol according to Formula I. In one aspect ofthe invention, the carbocyanine dyes of the invention are sulfonated oneor more times.

In addition, the dyes of the invention are substituted by one or morechemically reactive groups (RGM) or conjugated substances as describedbelow. In a preferred embodiment, the dye of the invention issubstituted by only one RGM.

Many embodiments of the compounds of the invention possess an overallelectronic charge. It is to be understood that when such electroniccharges are shown to be present, they are balanced by the presence ofappropriate counterions, which may or may not be explicitly identified.A biologically compatible counterion, which is preferred for someapplications, is not toxic in biological applications, and does not havea substantially deleterious effect on biomolecules. Where the compoundof the invention is positively charged, the counterion is typicallyselected from, but not limited to, chloride, bromide, iodide, sulfate,alkanesulfonate, arylsulfonate, phosphate, perchlorate,tetrafluoroborate, tetraarylboride, nitrate and anions of aromatic oraliphatic carboxylic acids. Where the compound of the invention isnegatively charged, the counterion is typically selected from, but notlimited to, alkali metal ions, alkaline earth metal ions, transitionmetal ions, ammonium or substituted ammonium or pyridinium ions.Preferably, any necessary counterion is biologically compatible, is nottoxic as used, and does not have a substantially deleterious effect onbiomolecules. Counterions are readily changed by methods well known inthe art, such as ion-exchange chromatography, or selectiveprecipitation.

It is to be understood that the dyes of the invention have been drawn inone or another particular electronic resonance structure. Every aspectof the instant invention applies equally to dyes that are formally drawnwith other permitted resonance structures, as the electronic charge onthe subject dyes is delocalized throughout the dye itself.

In one embodiment of the invention, the dye contains at least one L-RGM,where RGM is the reactive group that is attached to the dye by acovalent linkage L. In certain embodiments, the covalent linkageattaching the dye to RGM contains multiple intervening atoms that serveas a spacer. The dyes with a RGM label a wide variety of organic orinorganic substances that contain or are modified to contain functionalgroups with suitable reactivity, resulting in chemical attachment of theconjugated substance. As used herein, “reactive group moiety (RGM)”means moiety on the compound that is capable of chemically reacting witha functional group on a different compound to form a covalent linkage.Typically the reactive group is an electrophile or nucleophile that canform a covalent linkage through exposure to the corresponding functionalgroup that is a nucleophile or electrophile, respectively.Alternatively, the reactive group is a photoactivatable group, andbecomes chemically reactive only after illumination with light of anappropriate wavelength. Typically, the conjugation reaction between thereactive dye and the substance to be conjugated results in one or moreatoms of the reactive group RGM to be incorporated into a new linkage Lattaching the dye to the conjugated substance. Selected examples ofreactive groups and linkages are shown in Table 1 where the reaction ofan electrophilic group and a nucleophilic group yields a covalentlinkage.

TABLE 1 Examples of RGM groups that are used for preparing covalentlinkages: Electrophilic Group Nucleophilic Group Resulting Conjugateactivated esters* amines/anilines carboxamides acrylamides thiolsthioethers acyl azides** amines/anilines carboxamides acyl halidesamines/anilines carboxamides acyl halides alcohols/phenols esters acylnitriles alcohols/phenols esters acyl nitriles amines/anilinescarboxamides aldehydes amines/anilines imines aldehydes or ketoneshydrazines hydrazones aldehydes or ketones hydroxylamines oximes alkylhalides amines/anilines alkyl amines alkyl halides carboxylic acidsesters alkyl halides thiols thioethers alkyl halides alcohols/phenolsethers allcyl sulfonates thiols thioethers alkyl sulfonates carboxylicacids esters alkyl sulfonates alcohols/phenols ethers anhydridesalcohols/phenols esters anhydrides amines/anilines carboxamides arylhalides thiols thioethers aryl halides amines aryl amines aziridinesthiols thioethers boronates glycols boronate esters carbodiimidescarboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic acidsesters epoxides thiols thioethers haloacetamides thiols thioethershaloplatinate amino platinum complex haloplatinate heterocycle platinumcomplex haloplatinate thiol platinum complex halotriazinesamines/anilines aminotriazines halotriazines alcohols/phenols triazinylethers imido esters amines/anilines amidines isocyanates amines/anilinesureas isocyanates alcohols/phenols urethanes isothiocyanatesamines/anilines thioureas maleimides thiols thioethers phosphoramiditesalcohols phosphite esters silyl halides alcohols silyl ethers sulfonateesters amines/anilines alkyl amines sulfonate esters thiols thioetherssulfonate esters carboxylic acids esters sulfonate esters alcoholsethers sulfonyl halides amines/anilines sulfonamides sulfonyl halidesphenols/alcohols sulfonate esters *Activated esters, as understood inthe art, generally have the formula —COL, where L is a good leavinggroup (e.g. succinimidyloxy (—ONC₄H₄O₂) sulfosuccinimidyloxy(—ONC₄H₃O₂—SO₃H), -1-oxybenzotriazolyl (—OC₆H₄N₃); or an aryloxy groupor aryloxy substituted one or more times by electron withdrawingsubstituents such as nitro, fluoro, chloro, cyano, or trifluoromethyl,or combinations thereof, used to form activated aryl esters; or acarboxylic acid activated by a carbodiimide to form an anhydride ormixed anhydride —OCOAlk or —OCN(Alk₁)NH(Alk₂), where Alk₁ and Alk₂,which may be the same or different, are C₁-C₂₀ alkyl, C₁-C₂₀perfluoroalkyl, or C₁-C₂₀ alkoxy; or cyclohexyl, 3-dimethylaminopropyl,or N-morpholinoethyl). **Acyl azides can also rearrange to isocyanates.

Choice of the reactive group used to attach the dye to the substance tobe conjugated typically depends on the functional group on the substanceto be conjugated and the type or length of covalent linkage desired. Thetypes of functional groups typically present on the organic or inorganicsubstances include, but are not limited to, amines, amides, thiols,alcohols, phenols, aldehydes, ketones, phosphonates, imidazoles,hydrazines, hydroxylamines, disubstituted amines, halides, epoxides,carboxylate esters, sulfonate esters, purines, pyrimidines, carboxylicacids, olefinic bonds, or a combination of these groups. A single typeof reactive site may be available on the substance (typical forpolysaccharides), or a variety of sites may occur (e.g. amines, thiols,alcohols, phenols), as is typical for proteins. A conjugated substancemay be conjugated to more than one dye, which may be the same ordifferent, or to a substance that is additionally modified by a hapten,such as biotin. Although some selectivity can be obtained by carefulcontrol of the reaction conditions, selectivity of labeling is bestobtained by selection of an appropriate reactive dye.

Typically, RGM will react with an amine, a thiol, an alcohol, analdehyde or a ketone. Preferably RGM reacts with an amine or a thiolfunctional group. In one embodiment, RGM is an acrylamide, a reactiveamine (including a cadaverine or ethylenediamine), an activated ester ofa carboxylic acid (typically a succinimidyl ester of a carboxylic acid),an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, ananhydride, an aniline, an aryl halide, an azide, an aziridine, aboronate, a carboxylic acid, a diazoalkane, a haloacetamide, ahalotriazine, a hydrazine (including hydrazides), an imido ester, anisocyanate, an isothiocyanate, a maleimide, a phosphoramidite, areactive platinum complex, a sulfonyl halide, or a thiol group. By“reactive platinum complex” is particularly meant chemically reactiveplatinum complexes such as described in U.S. Pat. Nos. 5,580,990;5,714,327; 5,985,566.

Where the reactive group is a photoactivatable group, such as an azide,diazirinyl, azidoaryl, or psoralen derivative, the dye becomeschemically reactive only after illumination with light of an appropriatewavelength. Where RGM is an activated ester of a carboxylic acid, thereactive dye is particularly useful for preparing dye-conjugates ofproteins, nucleotides, oligonucleotides, or haptens. Where RGM is amaleimide or haloacetamide the reactive dye is particularly useful forconjugation to thiol-containing substances. Where RGM is a hydrazide,the reactive dye is particularly useful for conjugation toperiodate-oxidized carbohydrates and glycoproteins, and in addition isan aldehyde-fixable polar tracer for cell microinjection. Preferably,RGM is a carboxylic acid, a succinimidyl ester of a carboxylic acid, ahaloacetamide, a hydrazine, an isothiocyanate, a maleimide group, analiphatic amine, a perfluorobenzamido, an azidoperfluorobenzamido group,or a psoralen. More preferably, RGM is a succinimidyl ester of acarboxylic acid, a maleimide, an iodoacetamide, or a reactive platinumcomplex. Based on the above-mentioned attributes, the appropriatereactive dyes of the invention are selected for the preparation of thedesired dye-conjugates, whose advantageous properties make them usefulfor a wide variety of applications. Particularly useful dye-conjugatesinclude, among others, conjugates where substrate is a peptide, anucleotide, an antigen, a steroid, a vitamin, a drug, a hapten, ametabolite, a toxin, an environmental pollutant, an amino acid, aprotein, a nucleic acid, a nucleic acid polymer, a carbohydrate, alipid, an ion-complexing moiety, a glass or a non-biological polymer.Alternatively, substrate is a cell, a cellular system, a cellularfragment, or a subcellular particle (e.g. inter alia), a virus particle,a bacterial particle, a virus component, a biological cell (such asanimal cell, plant cell, bacteria, yeast, or protist), or a cellularcomponent. Reactive dyes typically label functional groups at the cellsurface, in cell membranes, organelles, or cytoplasm.

Typically substrate is an amino acid, a peptide, a protein, a tyramine,a polysaccharide, an ion-complexing moiety, a nucleoside, a nucleotide,an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, ahormone, a lipid, a lipid assembly, a polymer, a polymericmicroparticle, a biological cell or virus. More typically, substrate isa peptide, a protein, a nucleotide, an oligonucleotide, or a nucleicacid. When conjugating dyes of the invention to such biopolymers, it ispossible to incorporate more dyes per molecule to increase thefluorescent signal. For example, it is possible to incorporate at leastthree molecules of such dyes per molecule of antibody without loss oftotal fluorescence, whereas fluorescence of the spectrally comparableCy5 (wherein n=2) is strongly quenched when greater than approximatelytwo Cy5 dyes are incorporated per antibody. These results confirmproblems with Cy5 conjugates reported by others, e.g. BIOCONJUGATECHEM., 11, 696 (2000). The optimally labeled conjugates of the inventionare typically much more fluorescent than conjugates of the Cy5 dye or1,1′-crosslinked Cy5 at the same antibody concentration.

In one embodiment, substrate is an amino acid (including those that areprotected or are substituted by phosphonates, carbohydrates, or C₁ toC₂₅ carboxylic acids), or is a polymer of amino acids such as a peptideor protein. Preferred conjugates of peptides contain at least five aminoacids, more preferably 5 to 36 amino acids. Preferred peptides include,but are not limited to, neuropeptides, cytokines, toxins, proteasesubstrates, and protein kinase substrates. Preferred protein conjugatesinclude enzymes, antibodies, lectins, glycoproteins, histones, albumins,lipoproteins, avidin, streptavidin, protein A, protein G,phycobiliproteins and other fluorescent proteins, hormones, toxins,chemokines and growth factors. In one preferred aspect, the conjugatedprotein is a phycobiliprotein, such as allophycocyanin, phycocyanin,phycoerythrin, allophycocyanin B, B-phycoerythrin, andphycoerythrocyanin, (for example, see U.S. Pat. No. 5,714,386 toRoederer (1998)). Particularly preferred are conjugates ofR-phycoerythrin and of allophycocyanin with selected dyes of theinvention that serve as excited-state energy acceptors or donors. Inthese conjugates, excited state energy transfer results in longwavelength fluorescence emission when excited at relatively shortwavelengths.

In one aspect of the invention, substrate is a conjugated substance thatis an antibody (including intact antibodies, antibody fragments, andantibody sera, etc.), an amino acid, an angiostatin or endostatin, anavidin or streptavidin, a biotin (e.g. an amidobiotin, a biocytin, adesthiobiotin, etc.), a blood component protein (e.g. an albumin, afibrinogen, a plasminogen, etc.), a dextran, an enzyme, an enzymeinhibitor, an IgG-binding protein (e.g. a protein A, protein G, proteinA/G, etc.), a fluorescent protein (e.g. a phycobiliprotein, an aequorin,a green fluorescent protein, etc.), a growth factor, a hormone, a lectin(e.g. a wheat germ agglutinin, a conconavalin A, etc.), alipopolysaccharide, a metal-binding protein (e.g. a calmodulin, etc.), amicroorganism or portion thereof (e.g. a bacteria, a virus, a yeast,etc.), a neuropeptide and other biologically active factors (e.g. adermorphin, a deltropin, an endomorphin, an endorphin, a tumor necrosisfactor etc.), a non-biological microparticle (e.g. of ferrofluid, gold,polystyrene, etc.), a nucleotide, an oligonucleotide, a peptide toxin(e.g. an apamin, a bungarotoxin, a phalloidin, etc.), aphospholipid-binding protein (e.g. an annexin, etc.), a small-moleculedrug (e.g. a methotrexate, etc.), a structural protein (e.g. an actin, afibronectin, a laminin, a microtubule-associated protein, a tublin,etc.), or a tyramide.

In another embodiment, substrate is a nucleic acid base, nucleoside,nucleotide or a nucleic acid polymer, including those that are modifiedto possess an additional linker or spacer for attachment of the dyes ofthe invention, such as an alkynyl linkage (U.S. Pat. No. 5,047,519), anaminoallyl linkage (U.S. Pat. No. 4,711,955), or aheteroatom-substituted linker (U.S. Pat. No. 5,684,142) or otherlinkage. In another embodiment, the conjugated substance is a nucleosideor nucleotide analog that links a purine or pyrimidine base to aphosphate or polyphosphate moiety through a noncyclic spacer. In anotherembodiment, the dye is conjugated to the carbohydrate portion of anucleotide or nucleoside, typically through a hydroxyl group butadditionally through a thiol or amino group (U.S. Pat. Nos. 5,659,025;5,668,268; 5,679,785). Typically, the conjugated nucleotide is anucleoside triphosphate or a deoxynucleoside triphosphate or adideoxynucleoside triphosphate. Incorporation of methylene moieties ornitrogen or sulfur heteroatoms into the phosphate or polyphosphatemoiety is also useful. Nonpurine and nonpyrimidine bases such as7-deazapurines (U.S. Pat. No. 6,150,510) and nucleic acids containingsuch bases can also be coupled to dyes of the invention. Nucleic acidadducts prepared by reaction of depurinated nucleic acids with amine,hydrazide or hydroxylamine derivatives provide an additional means oflabeling and detecting nucleic acids, e.g. “A method for detectingabasic sites in living cells: age-dependent changes in base excisionrepair.” Atamna H, Cheung I, Ames B N. PROC. NATL. ACAD. SCI. U.S.A. 97,686-691 (2000).

Preferred nucleic acid polymer conjugates are labeled, single- ormulti-stranded, natural or synthetic DNA or RNA, DNA or RNAoligonucleotides, or DNA/RNA hybrids, or incorporate an unusual linkersuch as morpholine derivatized phosphates, or peptide nucleic acids suchas N-(2-aminoethyl)glycine units. When the nucleic acid is a syntheticoligonucleotide, it typically contains fewer than 50 nucleotides, moretypically fewer than 25 nucleotides. Conjugates of peptide nucleic acids(PNA) (Nielsen, et al. U.S. Pat. No. 5,539,082,) may be preferred forsome applications because of their generally faster hybridization rates.

In one embodiment, the conjugated oligonucleotides of the invention areaptamers for a particular target molecule, such as a metabolite, dye,hapten, or protein. That is, the oligonucleotides have been selected tobind preferentially to the target molecule. Methods of preparing andscreening aptamers for a given target molecule have been previouslydescribed and are known in the art [for example, U.S. Pat. No. 5,567,588to Gold (1996)].

In another embodiment, substrate is a carbohydrate that is typically apolysaccharide, such as a dextran, heparin, glycogen, amylopectin,mannan, inulin, starch, agarose and cellulose. Alternatively, thecarbohydrate is a polysaccharide that is a lipopolysaccharide. Preferredpolysaccharide conjugates are dextran, or lipopolysaccharide conjugates.

Conjugates having an ion-complexing moiety serve as indicators forcalcium, sodium, magnesium, zinc, potassium, or other biologicallyimportant metal ions. Preferred ion-complexing moieties are crown ethers(U.S. Pat. No. 5,405,975); derivatives of1,2-bis-(2-aminophenoxyethane)-N,N,N′,N′-tetraacetic acid (BAPTAchelators; U.S. Pat. Nos. 5,453,517; 5,516,911 and 5,049,673);derivatives of 2-carboxymethoxyaniline-N,N-di-acetic acid (APTRAchelators; AM. J. PHYSIOL., 256, C540 (1989)); or pyridine- andphenanthroline-based metal ion chelators (U.S. Pat. No. 5,648,270); orderivatives of nitrilotriacetic acid, see e.g. “Single-step synthesisand characterization of biotinylated nitrilotriacetic acid, a uniquereagent for the detection of histidine-tagged proteins immobilized onnitrocellulose”, McMahan SA and Burgess RR, ANAL. BIOCHEM., 236, 101-106(1996). Preferably, the ion-complexing moiety is a crown ether chelator,a BAPTA chelator, an APTRA chelator or a derivative of nitrilotriaceticacid.

Other conjugates of non-biological materials include dye-conjugates oforganic or inorganic polymers, polymeric films, polymeric wafers,polymeric membranes, polymeric particles, or polymeric microparticles(magnetic and non-magnetic microspheres); iron, gold or silverparticles; conducting and non-conducting metals and non-metals; andglass and plastic surfaces and particles. Conjugates are optionallyprepared by copolymerization of a dye that contains an appropriatefunctionality while preparing the polymer, or by chemical modificationof a polymer that contains functional groups with suitable chemicalreactivity. Other types of reactions that are useful for preparingdye-conjugates of polymers include catalyzed polymerizations orcopolymerizations of alkenes and reactions of dienes with dienophiles,transesterifications or transaminations. In another embodiment, theconjugated substance is a glass or silica, which may be formed into anoptical fiber or other structure. In one embodiment, conjugates ofbiological polymers such as peptides, proteins, oligonucleotides,nucleic acid polymers are also labeled with at least a secondluminescent dye, which is optionally an additional dye of the presentinvention, to form an energy-transfer pair. In some aspects of theinvention, the labeled conjugate functions as an enzyme substrate, andenzymatic hydrolysis disrupts the energy transfer. In another embodimentof the invention, the energy-transfer pair that incorporates a dye ofthe invention is conjugated to an oligonucleotide that displaysefficient fluorescence quenching in its hairpin conformation [theso-called “molecular beacons” of Tyagi, et al., NATURE BIOTECHNOLOGY,16, 49 (1998)] or fluorescence energy transfer.

The preparation of dye conjugates using reactive dyes is welldocumented, e.g. Hermanson G T, BIOCOJUGATE TECHNIQUES, Academic Press,New York (1996); Haugland R P, METHODS MOL. BIOL., 45, 205-21 (1995);and Brinkley, BIOCONJUGATE CHEM., 3, 2 (1992). Conjugates typicallyresult from mixing appropriate reactive dyes and the substance to beconjugated in a suitable solvent in which both are soluble. The majorityof the dyes of the invention are readily soluble in aqueous solutions,facilitating conjugation reactions with most biological materials. Forthose reactive dyes that are photoactivated, conjugation requiresillumination of the reaction mixture to activate the reactive dyes.

Synthesis

Synthesis of the cyanine dyes of the invention depends on initialpreparation of certain key intermediates. The intermediates have thefollowing general structures (for simplicity, all but a few of thepossible substituents are shown as hydrogen):

These basic structures are optionally further substituted, during orafter synthesis, to give the corresponding dye substituents as definedabove. For carbocyanines, the novel key intermediates are readilysynthesized by a reaction that is analogous to a Fischer indolesynthesis (see Sundberg RJ, THE CHEMISTRY OF INDOLES, Organic chemistry,a series of monographs, 1970, Academic Press). The typical synthesis ofdifferent substituted carbocyanines is illustrated in FIGS. 6-11.

Synthesis of the cyanine dyes of the invention, where RGM is at the3-position of the indolium and imidazolium, depends on initialpreparation of key intermediate IM 2. Licha, et al., U.S. Pat. No.6,083,485 (2000) described a typical synthesis of intermediate IM 2.These basic structures are optionally further substituted, during orafter synthesis, to give the corresponding dye substituents as definedabove. The novel key intermediates are readily synthesized by a reactionthat is analogous to a Fischer indole synthesis or through thecondensations of phenylenediamine with a carbonyl compound. The typicaltotal synthesis of 3-RGM-substituted carbocyanines is illustrated inFIG. 7.

Synthesis of the cyanine dyes of the invention, where attachment is atthe bridged and non-conjugated chain C, is either through the initialpreparation of key intermediate IM 4 or through the modification of thedisclosed procedures described for the synthesis of 1,1′-crosslinkedcarbocyanines (WO 01/02374 to Singh, et al). The typical total synthesisof carbocyanines with RGM on the non-conjugated chain C is illustratedin FIGS. 8 and 11.

Synthesis of the cyanine dyes of the invention, where attachment is atthe bridged and conjugated double bonds, depends on initial preparationof certain key bridged intermediates such as IM 5. For example,N,N′-diphenylformamidine, triethylorthoformate malonaldehydebis(phenylimine) hydrochloride, 1,1,3-trimethoxypropane,1,1,3,3-tetramethoxypropane and glutaconaldehyde dianil monochloride arethe well-known bridged intermediates used in the synthesis ofcarbocycanines. More examples of appropriate carbocyanines that havebridged and conjugated double bonds have been previously described inthe literature of U.S. Pat. No. 5,831,098 to Ollmann, Jr (1998); U.S.Pat. No. 6,086,737 to Patonay, et al. (2000); U.S. Pat. No. 6,048,982 toWaggoner (2000); and U.S. Pat. No. 5,453,505 to Lee, et al. (1995); U.S.Pat. No. 5,639,874 to Middendorf, et al. (1997); U.S. Pat. No. 3,864,644to Lincoln, et al. (1975); U.S. Pat. No. 4,011,086 to Simson (1977).Typically, each of R₁, R₂ and R₃ in Formula I, when present, ishydrogen. Where one of R₁, R₂ and R₃ is nonhydrogen, it is typically thesubstituent on the center carbon of BRIDGE. Similarly, where bridgedincorporates a 4-, 5-, or 6-membered ring, it typically occurs at thecenter of the bridged moiety. The typical total synthesis ofcarbocyanines substituted at the bridged and conjugated carbon atomswith RGM is illustrated in FIG. 9.

For the synthesis of carbocyanines, an appropriately substituted arylhydrazine (for simplicity, all but a few of the possible substituentsare shown as hydrogen), which is typically an appropriately substitutedphenylhydrazine, is reacted with an appropriately substituted methylketone to yield a 3,3-disubstituted 2-methylindole derivative (seeScheme 3). It is particularly suitable to utilize a sulfonatedphenylhydrazine derivative or a sulfonated naphthylhydrazine derivativeto increase the solubility of the final dye. The3,3-disubstituted-2-methylindole is then quaternized on the nitrogenatom to an indolium derivative with an alkylating agent that istypically an alkyl halide such as ethyl iodide, an alkylsulfonate suchas methyl p-toluenesulfonate or a cyclic sulfonate such aspropanesultone or butanesultone. Typically, the key indolium orbenzoindolium intermediates are sulfonated one or more times before orafter quaternization and subsequent condensation with the benzazoliummoiety and polymethine moiety to form the subject dyes. Variations onthese methods are well known in the art that yield substituents on thepolymethine bridge or on the indolium or benzolium portion of the dyeprecursor.

The azacarbocyanine dyes of the present invention can be analogouslysynthesized. [for example, see Leung W, et al., WO 02/26891; Brooker, etal., J. AM. CHEM. SOC., 64, 199 (1942); Heravi, et al., INDIAN J. CHEM.,36B, 1025 (1997); Smith, et al. SULFUR LETTERS, 17, 197 (1994);Chu-Moyer, et al. J. ORG. CHEM., 60, 5721 (1995); Turner, J. ORG. CHEM.,48, 3401 (1983); Couture, et al. J. HETEROCYCLIC CHEM., 24, 1765 (1987);Petric, et al. J. HETEROCYCLIC CHEM., 14, 1045, (1977); Barlin, et al.AUST. J. CHEM., 37, 1729 (1984); Saikachi et al. CHEM. & PHARM. BULL.,9, 941 (1961); Barlin, AUST. J. CHEM., 36, 983 (1983); Foye, et al., J.PHARM. SCI., 64, 1371 (1975); Khanna, et al. J. ORG. CHEM., 60, 960(1995)); British Patent No. 870,753 to Ficken, et al. (1961); Ficken, etal., “DIAZAINDENES AND THEIR QUANTERNARY SALTS-Part I”, pp 3202-3212(1959); Ficken, et al., “DIAZAINDENES AND THEIR QUANTERNARY SALTS-PartII”, pp 584-588 (1961)]. In general, the synthesis of these dyesrequires three precursors: the appropriate benzazolium or azabenzazoliumsalt (the “A” and “B” moieties), and a source for the polymethinespacer. Typically each component is selected so as to incorporate theappropriate chemical substituents, or functional groups (e.g. RGM) thatcan be converted to the appropriate substituents. The chemistry that isrequired to prepare and combine these precursors so as to yield any ofthe subject derivatives is generally well understood by one skilled inthe art.

It is recognized that there are many possible variations that may yieldequivalent results. The substituents on the aromatic carbons of theazabenzazolium moiety are typically incorporated in the parent aza- orpolyazabenzazole molecule prior to quaternization with an alkylatingagent. However, such substituents may also be incorporated during thesynthesis of the azabenzazole moiety. Alkyl, alkoxy, carboxyl, andhalogen substituents at aromatic carbons are typically already presentas substituents on the benzazole or azabenzazole precursors, or oncompounds that are readily converted to such precursors using methodswell-known in the art. Sulfonic acid groups are typically introduced onthe precursors prior to condensation of the cyanine dye [for example,see U.S. Pat. No. 5,767,287 to Bobrow, et al. (1998)]. Aminoalkyl groupstypically contain by a protecting group when they are first introduced,typically by substitution onto the benzazole or azabenzazole precursor.The protecting group is then removed after condensation of the cyaninedye. Aromatic amino groups are typically prepared via the reduction of anitro substituted benzazolium precursor, which in turn is prepared bythe nitration of the benzazole precursor.

The methods for synthesis of dyes that contain a variety of reactivegroups such as those described in Table 1 are well documented in theart. Particularly useful are amine-reactive dyes such as “activatedesters” of carboxylic acids, which are typically synthesized by couplinga carboxylic acid to a relatively acidic “leaving group”. Otherpreferred amine-reactive groups include sulfonyl halides, which areprepared from sulfonic acids using a halogenating agent such as PCl₅ orPOCl₃; halotriazines, which are prepared by the reaction of cyanurichalides with amines; and isocyanates or isothiocyanates, which areprepared from amines and phosgene or thiophosgene, respectively.

Dyes containing amines and hydrazides are particularly useful forconjugation to carboxylic acids, aldehydes and ketones. Most often theseare synthesized by reaction of an activated ester of a carboxylic acidor a sulfonyl halide with a diamine, such as cadaverine, or with ahydrazine. Alternatively, aromatic amines are commonly synthesized bychemical reduction of a nitroaromatic compound. Amines and hydrazinesare particularly useful precursors for synthesis of thiol-reactivehaloacetamides or maleimides by standard methods.

Nucleosides and nucleotides labeled with dyes of the invention areparticularly useful for some applications of nucleic acid labeling. Theuse of carbocyanine-amidites for labeling nucleotides and nucleosideshave been previously described [U.S. Pat. No. 5,986,086 to Brush, et al.(1999); U.S. Pat. No. 5,808,044 to Brush, et al. (1998); U.S. Pat. No.5,556,959 to Brush, et al. (1996)].

APPLICATIONS AND METHODS OF USE

In one aspect of the invention, the dye compounds of the invention areused to directly stain or label a sample so that the sample can beidentified or quantitated. For instance, such dyes may be added as partof an assay for a biological target analyte, as a detectable tracerelement in a biological or non-biological fluid; or for such purposes asphotodynamic therapy of tumors, in which a dyed sample is irradiated toselectively destroy tumor cells and tissues; or to photoablate arterialplaque or cells, usually through the photosensitized production ofsinglet oxygen. In one preferred embodiment, dye conjugate is used tostain a sample that comprises a ligand for which the conjugatedsubstance is a complementary member of a specific binding pair (e.g.Table 2).

Typically, the sample is obtained directly from a liquid source or as awash from a solid material (organic or inorganic) or a growth medium inwhich cells have been introduced for culturing, or a buffer solution inwhich cells have been placed for evaluation. Where the sample comprisescells, the cells are optionally single cells, including microorganisms,or multiple cells associated with other cells in two or threedimensional layers, including multicellular organisms, embryos, tissues,biopsies, filaments, biofilms, etc.

Alternatively, the sample is a solid, optionally a smear or scrape or aretentate removed from a liquid or vapor by filtration. In one aspect ofthe invention, the sample is obtained from a biological fluid, includingseparated or unfiltered biological fluids such as urine, cerebrospinalfluid, blood, lymph fluids, tissue homogenate, interstitial fluid, cellextracts, mucus, saliva, sputum, stool, physiological secretions orother similar fluids.

Alternatively, the sample is obtained from an environmental source suchas soil, water, or air; or from an industrial source such as taken froma waste stream, a water source, a supply line, or a production lot.

TABLE 2 Representative specific binding pairs Antigen Antibody BiotinAnti-biotin or avidin or streptavidin or neutravidin IgG* Protein A orprotein G or anti-IgG antibody Drug Drug receptor Toxin Toxin receptorCarbohydrate Lectin or carbohydrate receptor Peptide Peptide receptorNucleotide Complimentary nucleotide Protein Protein receptor Enzymesubstrate Enzyme DNA (RNA) aDNA (aRNA)** Hormone Hormone receptorPsoralen Nucleic acid Target molecule RNA or DNA aptamer Ion Ionchelator *IgG is an immunoglobulin; **aDNA and aRNA are the antisense(complementary) strands used for hybridization

In yet another embodiment, the sample is present on or in solid orsemi-solid matrix. In one aspect of the invention, the matrix is amembrane. In another aspect, the matrix is an electrophoretic gel, suchas is used for separating and characterizing nucleic acids or proteins,or is a blot prepared by transfer from an electrophoretic gel to amembrane. In another aspect, the matrix is a silicon chip or glassslide, and the analyte of interest has been immobilized on the chip orslide in an array (e.g. the sample comprises proteins or nucleic acidpolymers in a microarray). In yet another aspect, the matrix is amicrowell plate or microfluidic chip, and the sample is analyzed byautomated methods, typically by various methods of high-throughputscreening, such as drug screening.

The dye compounds of the invention are generally utilized by combining adye compound of the invention as described above with the sample ofinterest under conditions selected to yield a detectable opticalresponse. The term “dye compound” is used herein to refer to all aspectsof the claimed dyes, including both reactive dyes and dye conjugates.The dye compound typically forms a covalent or non-covalent associationor complex with an element of the sample, or is simply present withinthe bounds of the sample or portion of the sample. The sample is thenilluminated at a wavelength selected to elicit the optical response.Typically, staining the sample is used to determine a specifiedcharacteristic of the sample by further comparing the optical responsewith a standard or expected response.

A detectable optical response means a change in, or occurrence of, anoptical signal that is detectable either by observation orinstrumentally. Typically the detectable response is a change influorescence, such as a change in the intensity, excitation or emissionwavelength distribution of fluorescence, fluorescence lifetime,fluorescence polarization, or a combination thereof. The degree and/orlocation of staining, compared with a standard or expected response,indicates whether and to what degree the sample possesses a givencharacteristic. Some dyes of the invention may exhibit littlefluorescence emission, but are still useful as chromophoric dyes. Suchchromophores are useful as energy acceptors in FRET applications, or tosimply impart the desired color to a sample or portion of a sample.

For biological applications, the dye compounds of the invention aretypically used in an aqueous, mostly aqueous or aqueous-misciblesolution prepared according to methods generally known in the art. Theexact concentration of dye compound is dependent upon the experimentalconditions and the desired results, but typically ranges from about onenanomolar to one millimolar or higher. The optimal concentration isdetermined by systematic variation until satisfactory results withminimal background fluorescence are accomplished.

The dye compounds are most advantageously used to stain samples withbiological components. The sample may comprise heterogeneous mixtures ofcomponents (including intact cells, cell extracts, bacteria, viruses,organelles, and mixtures thereof), or a single component or homogeneousgroup of components (e.g. natural or synthetic amino acids, nucleicacids or carbohydrate polymers, or lipid membrane complexes). These dyesare generally non-toxic to living cells and other biological components,within the concentrations of use.

The dye compound is combined with the sample in any way that facilitatescontact between the dye compound and the sample components of interest.Typically, the dye compound or a solution containing the dye compound issimply added to the sample. Certain dyes of the invention, particularlythose that are substituted by one or more sulfonic acid moieties, tendto be impermeant to membranes of biological cells, and once insideviable cells are typically well retained. Treatments that permeabilizethe plasma membrane, such as electroporation, shock treatments or highextracellular ATP can be used to introduce selected dye compounds intocells. Alternatively, selected dye compounds can be physically insertedinto cells, e.g. by pressure microinjection, scrape loading, patch clampmethods, or phagocytosis.

Dyes that incorporate an aliphatic amine or a hydrazine residue can bemicroinjected into cells, where they can be fixed in place by aldehydefixatives such as formaldehyde or glutaraldehyde. This fixability makessuch dyes useful for intracellular applications such as neuronaltracing.

Dye compounds that possess a lipophilic substituent, such asphospholipids, will non-covalently incorporate into lipid assemblies,e.g. for use as probes for membrane structure; or for incorporation inliposomes, lipoproteins, films, plastics, lipophilic microspheres orsimilar materials; or for tracing. Lipophilic dyes are useful asfluorescent probes of membrane structure.

Chemically reactive dye compounds will covalently attach to acorresponding functional group on a wide variety of materials, formingdye conjugates as described above. Using dye compounds to label reactivesites on the surface of cells, in cell membranes or in intracellularcompartments such as organelles, or in the cell's cytoplasm, permits thedetermination of their presence or quantity, accessibility, or theirspatial and temporal distribution in the sample. Photoreactive dyes canbe used similarly to photolabel components of the outer membrane ofbiological cells or as photo-fixable polar tracers for cells.

Optionally, the sample is washed after staining to remove residual,excess or unbound dye compound. The sample is optionally combined withone or more other solutions in the course of staining, including washsolutions, permeabilization and/or fixation solutions, and solutionscontaining additional detection reagents. An additional detectionreagent typically produces a detectable response due to the presence ofa specific cell component, intracellular substance, or cellularcondition, according to methods generally known in the art. Where theadditional detection reagent has, or yields a product with, spectralproperties that differ from those of the subject dye compounds,multi-color applications are possible. This is particularly useful wherethe additional detection reagent is a dye or dye-conjugate of thepresent invention having spectral properties that are detectablydistinct from those of the staining dye.

The dye conjugates of the invention are used according to methodsextensively known in the art; e.g. use of antibody conjugates inmicroscopy and immunofluorescent assays; and nucleotide oroligonucleotide conjugates for nucleic acid hybridization assays andnucleic acid sequencing (e.g., U.S. Pat. No. 5,332,666 to Prober, et al.(1994); U.S. Pat. No. 5,171,534 to Smith, et al. (1992); U.S. Pat. No.4,997,928 to Hobbs (1991); and WO Appl. 94/05688 to Menchen, et al.).Dye-conjugates of multiple independent dyes of the invention possessutility for multi-color applications.

At any time after or during staining, the sample is illuminated with awavelength of light selected to give a detectable optical response, andobserved with a means for detecting the optical response. Equipment thatis useful for illuminating the dye compounds of the invention includes,but is not limited to, hand-held ultraviolet lamps, mercury arc lamps,xenon lamps, lasers and laser diodes. These illumination sources areoptionally integrated into laser scanners, fluorescence microplatereaders, standard or minifluorometers, or chromatographic detectors.Preferred embodiments of the invention are dyes that are be excitable ator near the wavelengths 633-636 nm, 647 nm, 660 nm, 680 nm and beyond700 nm, as these regions closely match the output of relativelyinexpensive excitation sources.

The optical response is optionally detected by visual inspection, or byuse of any of the following devices: CCD cameras, video cameras,photographic films, laser-scanning devices, fluorometers, photodiodes,quantum counters, epifluorescence microscopes, scanning microscopes,flow cytometers, fluorescence microplate readers, or by means foramplifying the signal such as photomultiplier tubes. Where the sample isexamined using a flow cytometer, examination of the sample optionallyincludes sorting portions of the sample according to their fluorescenceresponse.

One aspect of the instant invention is the formulation of kits thatfacilitate the practice of various assays using any of the dyes of theinvention, as described above. The kits of the invention typicallycomprise a colored or fluorescent dye of the invention, either presentas a chemically reactive label useful for preparing dye-conjugates, orpresent as a dye-conjugate where the conjugated substance is a specificbinding pair member, or a nucleoside, a nucleotide, an oligonucleotide,a nucleic acid polymer, a peptide, or a protein. The kit optionallyfurther comprises one or more buffering agents, typically present as anaqueous solution. The kits of the invention optionally further compriseadditional detection reagents, a purification medium for purifying theresulting labeled substance, luminescence standards, enzymes, enzymeinhibitors, organic solvent, or instructions for carrying out an assayof the invention.

EXAMPLES

Examples of some synthetic strategies for selected dyes of theinvention, as well as their characterization, synthetic precursors,conjugates and method of use are provided in the examples below. Furthermodifications and permutations will be obvious to one skilled in theart. The examples below are given so as to illustrate the practice ofthis invention. They are not intended to limit or define the entirescope of this invention.

Example 1 Preparation of Compound 1

The potassium salt of 2,3,3-trimethylindolinium-5-sulfonate issynthesized by Fisher indole synthesis through the reaction of4-hydrazinobenzenesulfonic acid and 3-methyl-2-butanone, followed byneutralizing the indolinyl sulfonic acid with saturated solution ofpotassium hydroxide in 2-propanol. The mixture of the potassium salt of2,3,3-trimethylindolinium-5-sulfonate (11 g, 39.7 mmol) and6-bromohexanoic acid (9.68 g, 49.6 mmol) in 1,2-dichlorobenzene (100 mL)is heated at 120° C. for 10 hours under nitrogen. The crude product istriturated with 2-propanol. The solid is filtered and washed with2-propanol and ether, and dried under vacuum to give Compound 1 (9.2 g).

Example 2 Preparation of Compound 2

To the solution of sodium ethoxide (173.4 mmol, prepared from 4.0 gsodium in 200 mL dry ethanol) is added ethyl 2-methylacetoacetate (25.0g, 173.4 mmol), followed by ethyl 6-bromohexanonate (44.5 g, 190.7mmol). The mixture is heated to reflux for 12 hours. After cooling toroom temperature, the mixture is filtered and the filtrate isconcentrated. The residue is treated with 1M HCl to pH 1 and the aqueoussolution is extracted with chloroform twice. The organic layer is washedwith brine and dried over Na₂SO₄. After removal of solvent, the residueis purified on silica gel to afford 15 g ethyl2-(5-ethoxycarbonyl)pentyl-2-methylacetoacetate.

The above acetoacetate (13.6 g) in methanol (130 mL) is mixed with asolution of NaOH (6.6 g) in water (60 mL). The mixture is stirred at 50°C. for 3 hours. After removal of methanol, the residue is acidified with1M HCl to pH 2. The aqueous solution is extracted with EtOAc (2×100 mL).The organic layer is washed with brine and dried over Na₂SO₄. The crudeproduct is purified with silica gel chromatography to yield7-methyl-8-oxo-nonanoic acid Compound 2 (6.4 g).

Example 3 Preparation of Compound 3

To the solution of Compound 2 (6.4 g) in methanol (50 mL) is added H₂SO₄(1.0 mL) dropwisely. The mixture is refluxed 30 min. After cooling toroom temperature, the reaction mixture is concentrated and the residueis diluted with ethyl acetate (100 mL). The solution is washed withsaturated NaHCO₃ and brine. The organic layer is dried over Na₂SO₄.After removal of solvent, the methyl 7-methyl-8-oxo-nonanoate Compound 3is obtained and used without further purification.

Example 4 Preparation of Compound 4

The mixture of 7-methyl-8-oxo-nonanoic acid (Compound 2, 4.2 g, 21.5mmol) and 4-hydrazinobenzenesulfonic acid (4.23 g, 22.5 mol) in aceticacid (30 mL) is heated to reflux for 8 hours. After removal of thesolvent, the residue is purified on silica gel to give Compound 4 (3.1g).

Example 5 Preparation of Compound 5

The mixture of methyl 7-methyl-8-oxo-nonanoate (Compound 3, 6.9 g, 34.4mmol) and 4-hydrazinobenzenesulfonic acid (6.45 g, 32.7 mol) in aceticacid (50 mL) is heated to reflux for 8 hours. After removal of thesolvent, the residue is purified on silica gel to give Compound 5 (9.7g).

Example 6 Preparation of Compound 6

A solution of Compound 4 (3.1 g) and potassium acetate (1.1 g) inmethanol (20 mL) is stirred at room temperature for 15 min. Afterremoval of methanol, the resulting potassium salt is heated with1,3-propanesultone (2.0 g) in 1,2-dichlorobenzene (5 mL) at 110° C. for1.5 hour. The mixture is cooled to room temperature and1,2-dichlorobenzene is decanted. The solid is triturated with 2-propanoland the free powder is filtered and washed with 2-propanol and ether anddried under vacuum to yield Compound 6.

Example 7 Preparation of Compound 7

A solution of Compound 5 (3.3 g) and potassium acetate (1.0 g) inmethanol (20 mL) is stirred at room temperature for 15 min. Afterremoval of methanol, the resulting potassium salt is heated with6-bromohexanoic acid (3.4 g) in 1,2-dichlorobenzene (10 mL) at 110° C.overnight. The mixture is cooled to room temperature and1,2-dichlorobenzene is decanted. The solid is triturated with ethylether and the free powder is filtered and washed with ether and driedunder vacuum to yield Compound 7.

Example 8 Preparation of Compound 8

A solution of Compound 4 (3.3 g) and potassium acetate (1.0 g) inmethanol (20 mL) is stirred at room temperature for 15 min. Afterremoval of methanol, the resulting potassium salt is heated with ethyl6-bromohexanonate (8.01 g) in 1,2-dichlorobenzene (10 mL) at 110° C.overnight. The mixture is cooled to room temperature and1,2-dichlorobenzene is decanted. The solid is triturated with ethylether and the free powder is filtered and washed with ether and driedunder vacuum to yield Compound 8.

Example 9 Preparation of Compound 9

The mixture of Boc-Lys(Boc)-OH (1.0 g, 2.9 mmol), N-hydroxysuccinimide(0.33 g, 2.9 mmol) and DCC (0.63 g, 3.03 mmol) in THF (25 mL) is stirredat room temperature overnight. After removal of solid, the filtrate[Boc-Lys(Boc)-OSu] is added to a solution of 6-aminocaproic acid (0.38g, 2.9 mmol) in water (20 mL), followed by addition of 2N Na₂CO₃ toadjust pH to 8-9. The mixture is stirred at room temperature overnight.After diluted with water (150 mL), the mixture is acidified with 4%aqueous HCl to pH 3 and extracted with ethyl acetate (2×50 mL). Thecombined extract is washed with brine and dried over Na₂SO₄. Afterremoval of solvent, the residue [Boc-Lys(Boc)-NH(CH₂)₅COOH] is dissolvedin 1,4-dioxane (15 mL), followed by addition of 4M HCl in dioxane (10mL). The mixture is stirred for 1 hour. The solvent is decanted and thesolid is washed with ethyl acetate (3×20 mL) and ether (3×20 mL). TheHCl salt of Compound 9 is dried under vacuum.

Example 10 Preparation of Compound 10

Compound 10 is prepared starting from DL-2, 3-diaminopropionic acidanalogously to the preparation of Compound 9.

Example 11 Preparation of Compound 11

5-Ethoxycarbonyl-2,3,3-trimethyl-3H-indole is synthesized through thereaction of ethyl 4-hydrazinobenzoate and 3-methyl-2-butanone. Compound11 is synthesized by the similar procedure described for the synthesisof Compound 1.

Example 12 Preparation of Compound 12

A solution of Compound 1 (100 mg, 0.283 mmol) and malonaldehydebis(phenylimine) monohydrochloride (77 mg, 0.297 mmol) in acetic acid(0.5 mL) and acetic anhydride (0.5 mL) is heated at 120° C. for 1 hour.The completion of the reaction is monitored by absorption spectra inmethanol. The solution of anyl intermediate is mixed with Compound 6(131 mg, 0.283 mol), then more acetic anhydride (0.5 mL) and pyridine(1.0 mL) is added. The mixture is heated for 30 min until the anylintermediate disappears (monitored by absorption spectra). The reactionmixture is cooled and poured into ethyl acetate (50 mL). The crudeproduct is collected by centrifugation and washed with ethyl acetatetwice. Preparative HPLC purification give Compound 12 as blue powder (35mg).

Example 13 Preparation of Compound 13

To a solution of Compound 12 (28.4 mg, 0.0334 mmol) andO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (26mg, 0.0864 mmol) in DMF (0.65 mL) is added triethylamine (0.04 mL). Themixture is stirred at room temperature for 1 h. The reaction mixture ispoured into EtOAc (15 mL). The di-succinimidyl ester of Compound 12 iscollected by centrifugation and washed with EtOAc (2×10 mL), EtOEt (1×10mL) and dried under vacuum.

The above di-succinimidyl ester of Compound 12 is dissolved in water (50mL) and a solution of Compound 9 (22.2 mg, 0.0667 mmol) in water (25 mL)[neutralized with Na₂CO₃ (7.1 mg, 0.0667 mmol)] is added slowly duringthe period of 30 minutes. The mixture is stirred at room temperatureovernight. After removal of solvent, the residue is purified bypreparative HPLC to give Compound 13 as blue powder (20 mg).

Example 14 Preparation of Compound 14

To a solution of Compound 13 (10 mg, 0.0093 mmol) in DMF (0.4 mL) isadded O—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(3.64 mg, 0.0121 mmol), followed by triethylamine (0.03 mL). The mixtureis stirred at room temperature for 1 h. The solution is poured intoEtOAc (15 mL). The solid is centrifuged and washed with EtOAc (3×10 mL),ether (1×10 mL) and dried under vacuum to give Compound 14 as brightblue powder (11 mg).

Example 15 Preparation of Compound 15

A solution of Compound 6 (100 mg, 0.217 mmol) and malonaldehydebis(phenylimine) monohydrochloride (56 mg, 0.217 mmol) in acetic acid(0.5 mL) and acetic anhydride (0.5 mL) is heated at 120° C. for 1 hour.The completion of the reaction is monitored by absorption spectra. Thesolution of anyl intermediate is mixed with Compound 7 (101 mg, 0.217mol), then more acetic anhydride (0.5 mL) and pyridine (1.0 mL) isadded. The mixture is heated for 30 min until the anyl intermediatedisappears (monitored by absorption spectra). The reaction mixture iscooled and poured into ethyl acetate (50 mL). The crude product iscollected by centrifugation and washed with ethyl acetate twice.Preparative HPLC purification gives Compound 15 as bright blue powder(15 mg).

Example 16 Preparation of Compound 16

To a solution of Compound 15 (6.0 mg, 0.0060 mmol) andO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (5.4mg, 0.018 mmol) in DMF (0.40 mL) is added triethylamine (0.04 mL). Themixture is stirred at room temperature for 1 h. The resulting solutionof di-succinimidyl ester of Compound 15 is diluted with DMF (30 mL),followed by addition of a solution of ethylenediamine (0.71 mg, 0.012mmol) in DMF (20 mL) during the period of 30 minutes. The mixture isstirred at room temperature overnight. After removal of solvent, theresidue is treated with 1N NaOH (2 mL). After the hydrolysis reaction iscompleting (monitored by HPLC), the reaction mixture is diluted withwater (5 mL) and neutralized with 1N HCl. Preparative HPLC purificationgives Compound 16 as blue powder (2 mg).

Example 17 Preparation of Compound 17

To a solution Compound 16 (2 mg, 0.002 mmol) in DMF (0.4 mL) is addedO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (0.8mg, 0.0027 mmol), followed by triethylamine (0.02 mL). The mixture isstirred at room temperature for 1 h. The solution is poured into EtOAc(15 mL). The solid is centrifuged and washed with EtOAc (3×10 mL), ether(1×10 mL) and dried under vacuum to give Compound 17 as bright bluepowder (2 mg).

Example 18 Preparation of Compound 18

A solution of Compound 1 (100 mg, 0.283 mmol) andN,N′-diphenylformamidine (58 mg, 0.297 mmol) in acetic acid (0.5 mL) andacetic anhydride (0.5 mL) is heated at 120° C. for 1 hour. Thecompletion of the reaction is monitored by absorption spectra inmethanol. The solution of anyl intermediate is mixed with Compound 6(131 mg, 0.283 mol), then more acetic anhydride (0.5 mL) and pyridine(1.0 mL) is added. The mixture is heated for 30 min until the anylintermediate disappears (monitored by absorption spectra). The reactionmixture is cooled and poured into ethyl acetate (50 mL). The crudeproduct is collected by centrifugation and washed with ethyl acetatetwice. Preparative HPLC purification gives Compound 18 (33 mg).

Example 19 Preparation of Compound 19

To a solution of Compound 18 (25 mg, 0.0303 mmol) andO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (24mg, 0.0788 mmol) in DMF (0.5 mL) is added triethylamine (0.04 mL). Themixture is stirred at room temperature for 1 h. The reaction mixture ispoured into EtOAc (15 mL). The di-succinimidyl ester of Compound 18 iscollected by centrifugation and washed with EtOAc (2×10 mL), EtOEt (1×10mL) and dried under vacuum.

The above di-succinimidyl ester of Compound 18 is dissolved in water (50mL) and a solution of Compound 10 (17.6 mg, 0.0606 mmol) in water (25mL) [neutralized with Na₂CO₃ (7.1 mg, 0.0606 mmol)] is added slowlyduring the period of 30 minutes. The mixture is stirred at roomtemperature overnight. After removal of solvent, the residue is purifiedby preparative HPLC to give Compound 19 (20 mg).

Example 20 Preparation of Compound 20

To a solution Compound 19 (10 mg, 0.0099 mmol) in DMF (0.4 mL) is addedO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (3.64mg, 0.0119 mmol), followed by triethylamine (0.03 mL). The mixture isstirred at room temperature for 1 h. The solution is poured into EtOAc(15 mL). The solid is centrifuged and washed with EtOAc (3×10 mL), ether(1×10 mL) and dried under vacuum to give Compound 20 (10 mg).

Example 21 Preparation of Compound 21

A solution of Compound 1 (100 mg, 0.283 mmol) and glutaconaldehydedianil hydrochloride (85 mg, 0.297 mmol) in acetic acid (0.5 mL) andacetic anhydride (0.5 mL) is heated at 120° C. for 1.5 hour. Thecompletion of the reaction is monitored by absorption spectra inmethanol. The solution of anyl intermediate is mixed with Compound 6(130 mg, 0.283 mol), then more acetic anhydride (0.5 mL) and pyridine(1.0 mL) is added. The mixture is heated for 30 min until the anylintermediate disappears (monitored by absorption spectra). The reactionmixture is cooled and poured into ethyl acetate (50 mL). The crudeproduct is collected by centrifugation and washed with ethyl acetatetwice. Preparative HPLC purification gives Compound 21 (20 mg).

Example 22 Preparation of Compound 22

To a solution of Compound 21 (20 mg, 0.0228 mmol) andO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (17.2mg, 0.0570 mmol) in DMF (0.5 mL) is added triethylamine (0.03 mL). Themixture is stirred at room temperature for 1 h. The reaction mixture ispoured into EtOAc (15 mL). The di-succinimidyl ester of Compound 21 iscollected by centrifugation and washed with EtOAc (2×10 mL), EtOEt (1×10mL) and dried under vacuum.

The above di-succinimidyl ester of Compound 21 is dissolved in water (40mL) and a solution of Compound 9 (15.2 mg, 0.0456 mmol) in water (25 mL)(neutralized with Na₂CO₃ (4.8 mg, 0.0456 mmol)) is added slowly duringthe period of 30 minutes. The mixture is stirred at room temperatureovernight. After removal of solvent, the residue is purified bypreparative HPLC to give Compound 22 (20 mg).

Example 23 Preparation of Compound 23

A solution of Compound 1 (100 mg, 0.283 mmol) and malonaldehydebis(phenylimine) monohydrochloride (77 mg, 0.297 mmol) in acetic acid(0.5 mL) and acetic anhydride (0.5 mL) is heated at 120° C. for 1 hour.The completion of the reaction is monitored by absorption spectra inmethanol. The solution of anyl intermediate is mixed with Compound 8(136 mg, 0.283 mol), then more acetic anhydride (0.5 mL) and pyridine(1.0 mL) is added. The mixture is heated for 30 min until the anylintermediate disappears (monitored by absorption spectra). The reactionmixture is cooled and poured into ethyl acetate (50 mL). The crudeproduct is collected by centrifugation and washed with ethyl acetatetwice. Preparative HPLC purification gives Compound 23 as bright bluepowder (30 mg).

Example 24 Preparation of Compound 24

To a solution of Compound 23 (30.0 mg, 0.0344 mmol) andO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (26.0mg, 0.0861 mmol) in DMF (0.60 mL) is added triethylamine (0.04 mL). Themixture is stirred at room temperature for 1 h. The resulting solutionof di-succinimidyl ester of Compound 23 is diluted with DMF (50 mL),followed by addition of a solution of ethylenediamine (4.1 mg, 0.0688mmol) in DMF (30 mL) during the period of 30 minutes. The mixture isstirred at room temperature overnight. After removal of solvent, theresidue is treated with 1N NaOH (3 mL). After the hydrolysis reaction iscompleting (monitored by HPLC), the reaction mixture is diluted withwater (10 mL) and neutralized with 1N HCl. Preparative HPLC purificationgives Compound 24 as blue powder (22 mg).

Example 25 Preparation of Compound 25

A solution of Compound 11 (100 mg, 0.292 mmol) and malonaldehydebis(phenylimine) monohydrochloride (79 mg, 0.306 mmol) in acetic acid(0.5 mL) and acetic anhydride (0.5 mL) is heated at 120° C. for 1 hour.The completion of the reaction is monitored by absorption spectra inmethanol. The solution of anyl intermediate is mixed with Compound 6(135 mg, 0.292 mol), then more acetic anhydride (0.5 mL) and pyridine(1.0 mL) is added. The mixture is heated for 30 min until the anylintermediate disappears (monitored by absorption spectra). The reactionmixture is cooled and poured into ethyl acetate (50 mL). The crudeproduct is collected by centrifugation and washed with ethyl acetatetwice. Preparative HPLC purification gives Compound 25 as bright bluepowder (33 mg).

Example 26 Preparation of Compound 26

To a solution of Compound 25 (30.0 mg, 0.0356 mmol) andO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (26.8mg, 0.0890 mmol) in DMF (0.60 mL) is added triethylamine (0.04 mL). Themixture is stirred at room temperature for 1 h. The resulting solutionof di-succinimidyl ester of Compound 25 is diluted with DMF (50 mL),followed by addition of a solution of ethylenediamine (4.3 mg, 0.0712mmol) in DMF (30 mL) during the period of 30 minutes. The mixture isstirred at room temperature overnight. After removal of solvent, theresidue is treated with 1N NaOH (3 mL). After the hydrolysis reaction iscompleting (monitored by HPLC), the reaction mixture is diluted withwater (10 mL) and neutralized with 1N HCl. Preparative HPLC purificationgives Compound 26 as blue powder (20 mg).

Example 27 Preparation of Compound 27

The reaction of di-potassium salt of1,1,2-trimethylbenzindolenium-6,8-disulfonic acid [BIOCONJUGATE CHEM.,356-362 (1996)] (5.0 g, 0.011 mmol) and 6-bromohexanoic acid (5.3 g,0.027 mmol) in dichlorobenzene at 120° C. overnight, followed by thesame work-up procedure as described for the synthesis of Compound 1,affords Compound 27 (4.5 g).

Example 28 Preparation of Compound 28

The Compound 28 is analogously synthesized by the same proceduredescribed for the synthesis of Compound 4 and Compound 6, starting fromthe reaction of 6-hydrazinonaphthalene 1,3-disulfonate [BIOCONJUGATECHEM., 356-362 (1996)] with 7-methyl-8-oxo-nonanoic acid Compound 2,followed by quaternization with 1,3-propanesultone.

Example 29 Preparation of Compound 29

A solution of Compound 27 (100 mg, 0.207 mmol) and malonaldehydebis(phenylimine) monohydrochloride (56 mg, 0.217 mmol) in acetic acid(0.5 mL) and acetic anhydride (0.5 mL) is heated at 120° C. for 1 hour.The completion of the reaction is monitored by absorption spectra inmethanol. The solution of anyl intermediate is mixed with Compound 28(123 mg, 0.207 mol), then more acetic anhydride (0.5 mL) and pyridine(1.0 mL) is added. The mixture is heated for 30 min until the anylintermediate disappears (monitored by absorption spectra). The reactionmixture is cooled and poured into ethyl acetate (50 mL). The crudeproduct is collected by centrifugation and washed with ethyl acetatetwice. Preparative HPLC purification gives Compound 29 as bright bluepowder (30 mg).

Example 30 Preparation of Compound 30

To a solution of Compound 29 (25 mg, 0.0225 mmol) andO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (17mg, 0.0562 mmol) in DMF (0.6 mL) is added triethylamine (0.03 mL). Themixture is stirred at room temperature for 1 h. The reaction mixture ispoured into EtOAc (15 mL). The di-succinimidyl ester of Compound 29 iscollected by centrifugation and washed with EtOAc (2×10 mL), EtOEt (1×10mL) and dried under vacuum.

The above di-succinimidyl ester of Compound 29 is dissolved in water (50mL) and a solution of Compound 9 (15 mg, 0.0450 mmol) in water (25 mL)[neutralized with Na₂CO₃ (4.8 mg, 0.0450 mmol)] is added slowly duringthe period of 30 minutes. The mixture is stirred at room temperatureovernight. After removal of solvent, the residue is purified bypreparative HPLC to give Compound 30 as blue powder (20 mg).

Example 31 Preparation of Compound 31

To a solution Compound 30 (10 mg, 0.0075 mmol) in DMF (0.4 mL) is addedO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (2.82mg, 0.0094 mmol), followed by triethylamine (0.03 mL). The mixture isstirred at room temperature for 1 h. The solution is poured into EtOAc(15 mL). The solid is centrifuged and washed with EtOAc (3×10 mL), ether(1×10 mL) and dried under vacuum to give Compound 31 as bright bluepowder (11 mg).

Example 32 Preparation of Compound 32

A solution of Compound 1 (353 mg, 1 mmol) and2-chloro-1-formyl-3-(hydroxymethylene)cyclohex-1-ene (173 mg, 1 mmol) in1-butanol (48 mL) and benzene (12 mL) is heated to reflux for 2 h. Afterthe reaction mixture is cooled to room temperature, a suspension ofCompound 6 (462 mg, 1 mmol) in 1-butanol (7 mL) and benzene (3 mL) isadded. The mixture is continued to reflux for 10 h with removal of waterby a Dean-Stark condenser. After removal of solvent, the residue ispurified by preparative HPLC to give Compound 32.

Example 33 Preparation of Compound 33

To a solution of Compound 32 (50.0 mg, 0.0574 mmol) andO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (43mg, 0.143 mmol) in DMF (1.0 mL) is added triethylamine (0.05 mL). Themixture is stirred at room temperature for 1 h. The resulting solutionof di-succinimidyl ester of Compound 32 is diluted with DMF (50 mL),followed by addition of a solution of ethylenediamine (6.9 mg, 0.115mmol) in DMF (30 mL) during the period of 30 minutes. The mixture isstirred at room temperature overnight. After removal of solvent, thechloro dye is converted to Compound 33 by 4-hydroxybenzoic acid andsodium hydride in DMF according to the procedure of N. Narayanan and G.Patonary (J. ORG. CHEM., 60, 2391 (1995)). Preparative HPLC purificationgives pure Compound 33 (20 mg).

Example 34 Preparation of Compound 34

To a solution of Compound 33 (10.0 mg, 0.01 mmol) andO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (8 mg,0.05 mmol) in DMF (0.5 mL) is added triethylamine (5 μL). The mixture isstirred at room temperature for 1 h, and precipitated with ether to givethe blue powder.

Example 35 Preparation of Compound 35

To Compound 20 in DMF is added 5 equivalents of anhydrous hydrazine. Themixture is stirred at ambient temperature for 15 minutes. The product isprecipitated with 4 volumes of ethyl acetate and purified by HPLC.

Example 36 Preparation of Compound 36

To Compound 14 in DMF at room temperature is added 4 equivalents oftriethylamine and 1.2 equivalents of N-(2-aminoethyl)maleimide,trifluoroacetic acid salt. The mixture is stirred at ambient temperaturefor 15 minutes. The product is precipitated with 4 volumes of ethylacetate and purified by HPLC.

Example 37 Preparation of Compound 37 (1,1′-crosslinked Cyanine)

Compound 37 is prepared from Compound 9 by modification of WO 01/02374(to R. Singh, et al.).

Example 38 Preparation of Compound 38 (1,1′-crosslinked Cyanine, SE)

Compound 37 is converted to Compound 38 analogous to the procedure ofCompound 14 as described in Example 14.

Example 39 Preparation of Compound 39

To a solution Compound 1 (5.0 g, 14.14 mmol) in DMF (20 mL) is addeddi(N-succinimidyl) carbonate (3.81 g, 14.85 mmol), followed bytriethylamine (3.9 mL, 228.29 mmol). The mixture is stirred at roomtemperature for 1 h. The solution is poured into EtOAc (150 mL). Thesolid is centrifuged and washed with EtOAc (3×100 mL), ether (1×100 mL)and dried under vacuum to give Compound 39, 6.0 g.

Example 40 Preparation of Compound 40

Compound 40 is prepared starting from Compound 6 analogously to thepreparation of Compound 39.

Example 41 Preparation of Compound 41

Compound 41 is prepared starting from Compound 7 analogously to thepreparation of Compound 39.

Example 42 Preparation of Compound 42

Compound 42 is prepared starting from Compound 8 analogously to thepreparation of Compound 39.

Example 43 Preparation of Compound 43

Compound 43 is prepared starting from Compound 27 analogously to thepreparation of Compound 39.

Example 44 Preparation of Compound 44

Compound 44 is prepared starting from Compound 28 analogously to thepreparation of Compound 39.

Example 45 Preparation of Compound 45

To a solution of Compound 39 (1.5 g, 3.33 mmol) in DMF (15 mL) is addedand t-BuOCONHCH₂CH(CH₂CH₂CH₂CH₂CH₂COOH)CH₂NH₂.HCl (product of AnaSpec,Inc.) (1.1 g, 3.33 mmol), followed by addition of triethylamine (0.34 g,0.46 mL, 3.33 mmol). The reaction mixture is stirred at room temperatureand the reaction is monitored by HPLC. After reaction is complete, thesolvent is removed and the residue is used for the next reaction withoutfurther purification.

Example 46 Preparation of Compound 46

The above Compound 45 is dissolved in TFA (10 mL) at 0° C. and thesolution is stirred at room temperature for 30 minutes. After removal ofTFA, the residue is treated with ethyl ether. The solid is collected byfiltration and washed with ether twice. After dried under vacuum, thesolid is dissolved in DMF (15 mL) and the solution is neutralized withtriethylamine. Then a solution of Compound 40 (1.86 g, 3.33 mmol) in DMF(10 mL) is added. The reaction mixture is stirred at room temperature.After the reaction is complete (monitored by HPLC), the solvent isremoved and the residue is treated with ethyl acetate to give Compound46.

Example 47 Preparation of Compound 47

Compound 47 is prepared starting from Compound 43 and Compound 44analogously to the preparation of Compound 46.

Example 48 Preparation of Compound 48

Compound 48 is prepared starting from Compound 40, BocNHCH₂CH₂NH₂ andCompound 41 analogously to the preparation of Compound 46.

Example 49 Preparation of Compound 49

Compound 49 is prepared starting from Compound 39, BocNHCH₂CH₂NH₂ andCompound 42 analogously to the preparation of Compound 46

Example 50 Preparation of Compound 50

Compound 46 (500 mg, 0.517 mmol) and malonaldehyde bis(phenylimine)monohydrochloride (67 mg, 0.258 mmol) are dissolved in acetic anhydride(3 mL), followed by addition of pyridine (3 mL). The mixture is heatedto 120° C. for 1 h. After cooling to room temperature, the mixture isdropped into ethyl acetate. The crude dye is collected by centrifugationand washed with ethyl acetate twice. Preparative HPLC purification givesCompound 50 as bright blue powder (200 mg).

Example 51 Preparation of Compound 51

Compound 46 (500 mg, 0.517 mmol) and N,N-diphenylformamidine (51 mg,0.258 mmol) are dissolved in acetic anhydride (3 mL), followed byaddition of pyridine (3 mL). The mixture is heated to 120° C. for 1 h.After cooling to room temperature, the mixture is dropped into ethylacetate. The crude dye is collected by centrifugation and washed withethyl acetate twice. Preparative HPLC purification gives Compound 51(220 mg).

Example 52 Preparation of Compound 52

Compound 46 (500 mg, 0.517 mmol) and glutaconaldehyde dianilhydrochloride (74 mg, 0.258 mmol) are dissolved in acetic anhydride (3mL), followed by addition of pyridine (3 mL). The mixture is heated to120° C. for 1 h. After cooling to room temperature, the mixture isdropped into ethyl acetate. The crude dye is collected by centrifugationand washed with ethyl acetate twice. Preparative HPLC purification givesCompound 52 (190 mg).

Example 53 Preparation of Compound 53

Compound 47 (500 mg, 0.407 mmol) and malonaldehyde bis(phenylimine)monohydrochloride (53 mg, 0.204 mmol) are dissolved in acetic anhydride(3 mL), followed by addition of pyridine (3 mL). The mixture is heatedto 120° C. for 1 h. After cooling to room temperature, the mixture isdropped into ethyl acetate. The crude dye is collected by centrifugationand washed with ethyl acetate twice. Preparative HPLC purification givesCompound 53 as bright blue powder (180 mg).

Example 54 Preparation of Compound 54

Compound 48 (500 mg, 0.525 mmol) and N,N-diphenylformamidine (51.5 mg,0.262 mmol) are dissolved in acetic anhydride (3 mL), followed byaddition of pyridine (3 mL). The mixture is heated to 120° C. for 1 h.After cooling to room temperature, the mixture is dropped into ethylacetate. The crude dye is collected by centrifugation and washed withethyl acetate twice and then dissolved in 1N NaOH (10 mL). After thehydrolysis reaction is completing (monitored by HPLC), the reactionmixture is diluted with water (10 mL) and neutralized with 1N HCl.Preparative HPLC purification gives Compound 54 (180 mg).

Example 55 Preparation of Compound 55

Compound 49 (500 mg, 0.582 mmol) and N,N-diphenylformamidine (57 mg,0.291 mmol) are dissolved in acetic anhydride (3 mL), followed byaddition of pyridine (3 mL). The mixture is heated to 120° C. for 1 h.After cooling to room temperature, the mixture is dropped into ethylacetate. The crude dye is collected by centrifugation and washed withethyl acetate twice and then dissolved in 1N NaOH (10 mL). After thehydrolysis reaction is completing (monitored by HPLC), the reactionmixture is diluted with water (10 mL) and neutralized with 1N HCl.Preparative HPLC purification gives Compound 55 (180 mg).

Example 56 Preparation of a Peptide-dye Conjugate

To aminophalloidin (3.5 mg, 4 μmol, Alexis Corp.) and the succinimidylester derivative Compound 14 (6.0 mg, 5 μmol) in DMF is addedN,N-diisopropylethylamine (2 μL, 11 μmol). The mixture is stirred atroom temperature for 3 hours. To this solution is added 7 mL of diethylether. The solid is collected by centrifugation. The crude product ispurified on SEPHADEX LH-20, eluting with water, followed by preparativeHPLC to give the pure phalloidin conjugate. The product is all effectivestain for F-actin filaments in fixed-cell preparations.

Example 57 Preparation of a Drug-dye Conjugate

A fluorescent dopamine D₂ antagonist is prepared as follows: To 10 mg ofN-(p-aminophenethyl)spiperone (Amlaiky, et al., FEBS LETT., 176, 436(1984)), and 10 μL N,N-diisopropylethylamine in 1 mL of DMF is added 15mg of Compound 14 or 20. After 3 hours, the reaction mixture is pouredinto 5 mL ether. The precipitate is centrifuged, then purified bychromatography on silica gel using 10-30% methanol in chloroform.

Example 58 Preparation of Protein-dye Conjugates

A series of dye conjugates of goat anti-mouse IgG (GAM), goatanti-rabbit IgG (GAR), streptavidin, transferrin and other proteins,including R-phycoerythrin (R-PE) and allophycocyanin (APC) are preparedby standard means (Haugland, et al., METH. MOL. BIOL., 45, 205 (1995);Haugland, METH. MOL. BIOL., 45, 223 (1995); Haugland, METH. MOL. BIOL.,45, 235 (1995); Haugland, CURRENT PROTOCOLS IN CELL BIOLOGY,16.5-1-16.5.22 (2000)) using Compound 14 or 20 and a mono-succinimidylester derivative of the Cy5 dye (Amersham Biosciences).

The typical method for protein conjugation with succinimidyl esters ofthe invention is as follows. Variations in ratios of dye to protein,protein concentration, time, temperature, buffer composition and othervariables that are well known in the art are possible that still yielduseful conjugates. A solution of the protein is prepared at about 10mg/mL in 0.1 M sodium bicarbonate. The labeling reagents are dissolvedin a suitable solvent such as DMF or DMSO at about 10 mg/mL. Water is asuitable solvent for many dyes of the invention. Predetermined amountsof the labeling reagents are added to the protein solutions withstirring. A molar ratio of 10 equivalents of dye to 1 equivalent ofprotein is typical, though the optimal amount varies with the particularlabeling reagent, the protein being labeled and the protein'sconcentration, and is determined empirically.

When optimizing the fluorescence yield and determining the effect ofdegree of substitution (DOS) on this brightness, it is typical to varythe ratio of reactive dye to protein over a several-fold range. Thereaction mixture is incubated at room temperature for one hour or on icefor several hours. The dye-protein conjugate is typically separated fromfree unreacted reagent by size-exclusion chromatography, such as onAmersham PD-10 resin equilibrated with phosphate-buffered saline (PBS).The initial, protein-containing colored band is collected and the degreeof substitution is determined from the absorbance at the absorbancemaximum of each fluorophore, using the extinction coefficient of thefree fluorophore. The dye-protein conjugate thus obtained can besubfractionated to yield conjugates with higher, lower or more uniformDOS.

Following is a specific example of using Compound 14 to prepare IgG-dyeconjugate:

-   Step 1. Preparing protein solution (Solution A): Mix 50 μL of 1 M    NaHCO₃ with 450 μL of IgG protein solution (4 mg/mL) to give 0.5 mL    protein sample solution. The resulted solution should have pH    8.5±0.5.-   Step 2. Preparing dye solution (Solution B): To 50 μL of DMSO add 1    mg of Compound 14, and stir until the compound is completely    dissolved.-   Step 3. Running conjugation reaction: Add the protein solution (A)    to the dye solution (B) with effective stirring or shaking, and keep    the reaction mixture stirred or shaken for 1-3 hrs.-   Step 4. Purifying the conjugate: a). Dilute 10× elution buffer with    de-ionized water to give 1× elution buffer (Solution C) that is used    to elute the protein conjugate from PD-10 column; b). Load the    column with the reaction mixture (from step 3, filtrated if    necessary) or supernatant as soon as the liquid in the pre-packed    column runs just below the top surface; c). Add 1 mL of the 1×    elution buffer as soon as the sample runs just below the top resin    surface; Repeat this ‘sample washing’ process twice; Add more 1×    elution buffer solution to elute the desired sample; d). Collect the    faster-running band that is usually the desired labeled protein.    Keep the slower-running band that is usually free or hydrolyzed dye    until the desired product is identified.-   Step 5. Characterizing the desired dye-protein conjugate: a).    Measure OD (absorbance) at 280 nm and 650 nm (Note: for most    spectrophotometers, the sample (from the column fractions) need be    diluted with de-ionized water so that the OD values are in the range    0.1 to 0.9). The O.D. (absorbance) 280 nm is the maximum absorption    of protein while 650 nm is the maximum absorption of Compound 14    amide (Note: to obtain accurate DOS, you must make sure that the    conjugate is free of the non-conjugated dye); b). Calculating DOS    using the following equation:    DOS=[dye]/[protein]=A ₆₅₀×ε_(p)/250000(A ₂₈₀−0.05A ₆₅₀)-    [dye] is the dye concentration, and can be readily calculated from    the Beer-Lambert Law: A=ε_(dye)C×L; [protein] is the target protein    concentration. This value can be either estimated by the weight    (added to the reaction) if the conjugation efficiency is high enough    (preferably >70%) or more accurately calculated by the Beer-Lambert    Law: A=ε_(protein)C×L. For example, IgG has the value to be 203,000    cm⁻¹M⁻¹. For effective labeling, the degree of substitution should    fall between 2-6 moles of Compound 14 to one mole of antibody.

Example 59 Fluorescent Labeling of Periodate-oxidized Proteins

Two samples of 5 mg each of goat IgG antibody in 1 mL of 0.1 M acetate,0.135 M NaCl, pH 5.5, are treated with 2.1 mg of sodium metaperiodate onice, for 1 and 2 hours, respectively. The reactions are stopped byaddition of 30 μL ethylene glycol. The antibodies are purified on aSephadex G25 column packed in PBS pH 7.2. One-tenth volume of 1 M sodiumbicarbonate is added to raise the pH and Compound 35 is added at a molarratio of dye to protein of 50:1. The reaction is stirred for 2 hours atroom temperature. Sodium cyanoborohydride is added to a finalconcentration of 10 mM and the reaction is stirred for 4 hours at roomtemperature. The antibody conjugates are purified by dialysis and onSephadex G25 columns as described above. Antibodies that are oxidizedfor 1 hour typically yield a degree of substitution of 1 mole of dye permole of IgG. Antibodies that are oxidized for 2 hours typically yield aDOS of approximately 2 mole of dye per mole of IgG. Periodate-oxidizedproteins in gels and on blots can also be labeled, essentially asdescribed in Estep TN and Miller TJ, (ANAL. BIOCHEM., 157, 100-105(1986)). The conjugates of Compound 35 exhibit greater fluorescence thanthe conjugates of Cy3 dye at similar DOS when conjugated to a widevariety of proteins.

Example 60 Labeling Beta-galactosidase with a Thiol-reactive Dye

A solution of beta-galactosidase, a protein rich in free thiol groups,is prepared in PBS (2.0 mg in 400 μL). The protein solution is thentreated with a 20 mg/L solution of the maleimide derivative Compound 36in DMF. Unreacted dye is removed on a spin column. The degree ofsubstitution by the dye is estimated using the extinction coefficient ofthe free dye as described in Example 58. The protein concentration isestimated from the absorbance at 280 nm, corrected for the absorbance ofCompound 36 at that wavelength.

Example 61 Total Fluorescence of Selected Dye-protein ConjugatesCompared with Cy5

In general, the higher the DOS, the brighter the Compounds 14 and 17bioconjugates relative to the Cy5 bioconjugates, although, Compound 14and 17 bioconjugates are brighter at all DOS tested. The decrease in theRQY of the Cy5 bioconjugates is found to be accompanied by an increasein the 600-nm absorbance band relative to the 650-nm absorbance band.The increase in extinction of the 600 nm band is always associated witha large quenching of the fluorescence. This result is completelysupportive of the work of Gruber, et al. (BIOCONJUGATE CHEM., 11, 696(2000)) who observed a similar correlation of an increased absorbance at600 nm and a large decrease in fluorescence intensity. FIG. 4 shows adirect comparison of the fluorescence emission of the Compound 14conjugate of GAR IgG at nearly equivalent DOS. The 600 nm absorbanceband is always much lower in extinction for Compound 14 than for anequivalently labeled Cy5 derivative. This general observation has nowbeen confirmed with several other proteins.

Example 62 Comparison of the Protein Conjugates Prepared from1,1′-crosslinked and Non-crosslinked “Cy5-like” Isomers with Compound 14

1,1′-Crosslinked Cy5 isomer is synthesized as described in Example 37and conjugated to GAR at various DOS. FIG. 4 is a direct comparison offluorescence properties of GAR conjugates prepared from Cy5 SE,Compounds 14 and 38. One can see that the 1,3′-intramolecularcrosslinking has resulted in a drastic improvement of fluorescenceperformance of Compound 14 GAR conjugates over those of Cy5(non-crosslinked cyanine) and Compound 38 (1,1′-crosslinked cyanine).Compound 14 GAR conjugate also has much weaker absorbance around 600 nm(non-fluorescent excitation). The brighter fluorescence emission ofcompound 14 GAR conjugate (than Cy5 and Compound 38) is observed at allof the tested DOS's.

Example 63 Comparison of the Fluorescence of Goat Anti-mouse IgG (GAM)

Conjugates of Cy3 and Compound 20 are prepared analogously to theprocedure of Example 58 with Compound 20 and the Cy3 reactive dyes at avariety of degrees of substitution ranging from 1.0-12. The conjugatesare characterized using excitation wavelength=532 nm analogously toExample 58.

Example 64 The Photostability of Compound 13 is Greater than that of Cy5Free Acid

Photobleaching experiments are performed at 0.1 μM concentrations ofCompound 13 and commercially available Cy5 free acid. Both of thecompounds are irridated with A100 W Mercury lamp in PBS (pH 7.0), whereboth of the dyes receive the same amount of irradiation as determined byphotometric measurements. As shown in FIG. 5, Compound 13 remains about3 times brighter than the Cy5 free acid after 500 minutes ofillumination.

Example 65 Fluorescence Energy Transfer in Conjugates of R-phycoerythrinand Allophycocyanin

R-phycoerythrin (R-PE) conjugate of Compound 14 or 17 is prepared as inExample 58 with a DOS sufficiently high to quench the donor fluorescencealmost completely (DOS about 4-8). The resulting phycobiliproteinconjugate is excited at 488 nm and the fluorescence emission is comparedto that of unmodified R-phycoerythrin excited at the same wavelength.Highly efficient energy transfer (>99%) occurs from the protein to thefluorescent dye. A conjugate of these complexes with streptavidin isprepared essentially as described by Haugland (METH. MOL. BIOL., 45, 205(1995)). This streptavidin conjugate retains the energy transferproperties and is useful for cell staining in flow cytometers thatutilize the argon-ion laser for excitation. Tandem conjugates ofallophycocyanin can also be made, with longer wavelength dyes of theinvention such as Compound 34 yield emission well beyond 700 nm whenexcited near 633 nm.

Example 66 Labeling of Actin in Cultured Mammalian Cells

Bovine pulmonary artery cells (BPAEC) are grown to 30-50% of confluenceon glass. The cells are fixed with 3.7% formaldehyde, permeabilized with0.2% Triton X-100, and blocked with 6% BSA. The cells are incubated withthe phalloidin dye-conjugate of Example 56. The cells are rinsed withblocking buffer and mounted in PBS pH 7.4. The stained cells displayactin filaments decorated with red fluorescence.

Example 67 Preparation and Use of a Fluorescent Alpha-bungarotoxinDye-conjugate

Alpha-Bungarotoxin (1 mg) in 25 μL 0.1 M NaHCO₃ is treated with 1.5equivalents of Compound 14 or 20 at room temperature for 2 hours. Theproduct is purified by size exclusion, by ion exchange chromatography,and finally by reverse-phase HPLC. The conjugate is used for staining ofacetylcholine receptors.

Example 68 Preparation and Use of a Fluorescent Tyramide

A 2-fold molar excess of tyramine hydrochloride is added to Compound 20in aqueous solution at room temperature followed by an excess oftriethylamine. After 30 minutes the red solid is precipitated withacetone, washed with ether and purified by preparative HPLC. Bovinepulmonary artery cells (BPAEC) are grown to 30-50% of confluence onglass. The cells are fixed with 3.7% formaldehyde, permeabilized with0.2% Triton X-100, and blocked with 1 mg/mL streptavidin and 1 mMbiotin. After washing, cells are exposed to about 0.05 μg/mL ofbiotinylated anti-cytochrome C oxidase (anti-COX) then incubated withStreptavidin-HRP conjugate at room temperature. Cells are rinsed again.The sample is then incubated with Compound 20 tyramide and examinedusing fluorescence microscopy.

Example 69 Preparation of Aminodextran Dye-conjugates

70,000-MW aminodextran (50 mg) derivatized with an average of 13 aminogroups is dissolved at 10 mg/mL in 0.1 M NaHCO₃. Compound 14 or 20 or 31is added so as to give a dye/dextran ratio of about 10-15. After 6-12hours the conjugate is purified on SEPHADEX G-50, eluting with water.Typically 4-6 moles of dye are conjugated to 70,000 MW dextran.

Example 70 Preparation of Fluorescent-dye Labeled Microspheres

Uniform microspheres are chemically modified to have functional groupssuch as amino or carboxyl or aldehydes. These functionalizedmicrospheres are covalently conjugated with the corresponding reactivedyes as listed in Table 1. For example, the amine-modified microspheresare readily conjugated to the dyes of the invention through succinimidylesters such as Compounds 14, 17, 20 and 31. A dye-labeled protein iscovalently coupled through its amine residues to the carboxylate groupsof the polymer using ethyl 3-(dimethylaminopropyl)carbodiimide (EDAC).

The dyes of invention can also be physically adsorbed on microspheres.For example, carboxylate-modified microspheres are suspended in asolution of a protein that has been conjugated to a dye of theinvention. The protein is passively adsorbed on the microspheres, andexcess protein is removed by centrifugation and washing. Microparticlesof a size that cannot be centrifuged are separated from excess proteinby dialysis through a semi-permeable membrane with a high MW cutoff orby gel filtration chromatography. Another example is that biotinylatedmicrospheres are treated with a streptavidin, avidin or anti-biotinconjugate of a dye of the invention.

Example 71 Preparation of Fluorescent Liposomes Using Dyes of theInvention

Selected dyes of the invention (such as Compound 13 and 19) aresufficiently water soluble to be incorporated into the interior ofliposomes by methods well known in the art (J. BIOL. CHEM., 257, 13892(1982) and PROC. NATL. ACAD. SCI., USA 75, 4194 (1978)). Alternatively,liposomes containing dyes of the invention having a lipophilicsubstituent (e.g. alkyl having 11-22 carbons), within their membranesare prepared by co-dissolving the fluorescent lipid and the unlabeledlipids phospholipid(s) that make up the liposome before forming theliposome dispersion essentially as described by Szoka Jr., et al. (ANN.REV. BIOPHYS. BIOENG., 9, 467 (1980)).

Example 72 Preparation of Dye-bacteria Conjugates

Heat-killed Escherichia coli are suspended at 10 mg/mL in pH 8-9 bufferthen incubated with 0.5-1.0 mg/mL of an amine-reactive dye, typically asuccinimidyl ester derivative (such as Compound 14 or 20 or 31). After30-60 minutes the labeled bacteria are centrifuged and washed severaltimes with buffer to remove any unconjugated dye. Labeled bacteria isanalyzed by flow cytometry.

Example 73 Preparation of Nucleotide-dye Conjugates

To 2 mg of 5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphate (SigmaChemical) in 100 μL water is added Compound 14 or 20 in 100 μL DMF and 5μL triethylamine. After 3 hours, the solution is evaporated and theresidue is purified by HPLC. The product fractions are lyophilized togive the red-fluorescent nucleotide conjugate. Alternatively,fluorescent dye-conjugates of deoxyuridine 5′-triphosphate are preparedfrom 5-(3-amino-1-propynyl)-2′-deoxyuridine 5′-triphosphate, or bytreating a thiolated nucleotide or a thiophosphate nucleotide with athiol-reactive dye of the invention (such as the maleimide Compound 36).Additionally, 2′-(or 3′)-2-aminoethylaminocarbonyladenosine5′-triphosphate is reacted with a slight excess of Compound 14 and,following precipitation with ethanol, the ribose-modified product ispurified by preparative HPLC. Additional nucleotides conjugated with thedyes of invention can be readily prepared by someone skilled in the artfollowing the published procedures such as Nimmakayalu M, et al.,BIOTECHNIQUES, 28, 518-522 (2000); Muhlegger K, et al., BIOL. CHEM.HOPPE SEYLER, 371, 953-965 (1990); Giaid A, et al. HISTOCHEMISTRY, 93,191-196 (1989).

Example 74 Preparation of an Oligonucleotide Dye-conjugate

A 5′-amine-modified, 18-base M13 primer sequence (about 100 μg) isdissolved in 4 μL water. To this is added 250 μg of Compound 14 or 20 in100 μL 0.1 M sodium borate, pH 8.5. After 16 hours, 10 μL of 5 M NaCland 3 volumes of cold ethanol are added. The mixture is cooled to −20°C., centrifuged, the supernatant is decanted, the pellet is rinsed withethanol and then dissolved in 100 μL water. The labeled oligonucleotideis purified by HPLC. The desired peak is collected and evaporated togive the fluorescent oligonucleotide.

Example 75 In situ Hybridization of an RNA Probe

Mouse fibroblasts are fixed and prepared for mRNA in situ hybridizationusing standard procedures. A dye-labeled RNA probe is prepared by invitro transcription of a plasmid containing the mouse actin structuralgene cloned downstream of a phage T3 RNA polymerase promoter. Labelingreactions comprise combining 2 μL DNA template (1 μg DNA), 1 μL each of10 mM ATP, CTP and GTP, 0.75 μL 10 mM UTP, 2.5 μL 1 mMaminoallyl-labeled UTP, 2 μL 10× transcription buffer (400 mM Tris, pH8.0, 100 mM MgCl₂, 20 mM spermidine, 100 mM NaCl), 1 μL T3 RNApolymerase (40 units/μL), 1 μL 2 mg/mL BSA, and 8.75 μL water. Reactionsare incubated at 37° C. for two hours. The DNA template is removed bytreatment with 20 units DNase I for 15 minutes, at 37° C. The RNAtranscript is purified by extraction with an equal volume ofphenol:chloroform, 1:1, then by chromatography on SEPHADEX G50. LabeledRNA is denatured for 5 minutes at 50° C., then hybridized to cellularpreparations using standard procedures. The long-wavelength fluorescenceof the labeled cells is detected by excitation through an optical filteroptimized for Cy5-like dyes.

Example 76 Preparing DNA Hybridization Probes Using Amine-modified DNAand an Amine-reactive Dye of the Invention

Nick translation is performed using pUC1.77 plasmid DNA containing achromosome 1 human alpha-satellite probe. To a microcentrifuge tube isadded, in the following order: 23.5 μL water, 5 μL 10× Nick Translationbuffer (0.5 M Tris-HCl, 50 mM MgCl₂, 0.5 mg/mL BSA, pH 7.8), 5 μL 0.1 MDTT, 4 μL d(GAC)TP mix (0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP), 1 μL 0.5mM dTTP, 4 μL 0.5 mM aminoallyl-dUTP, 1 μL 1 μg/μL template DNA, 5 μLDNase I (1 μg/mL, 2000 Kunitz units/mg), 1.5 μL DNA polymerase I (10U/μL). The tube is incubated 2 hours at 15° C., then brought to a finalvolume of 100 μL with water. The amine-modified DNA is purified using aQIAQUICK PCR purification Kit (Qiagen). The amine-modified DNA isresuspended in 5 μL water. To the solution is added 3 μL 25 mg/mL sodiumbicarbonate and 50 μg of Compound 14 or 20 in 5 μL DMF. The reaction isincubated for 1 hour at room temperature in the dark, to the reaction isadded 90 μL water, and it is purified using a QIAQUICK PCR purificationkit (Qiagen). The labeled DNA products are suitable for in situhybridization experiments, use on microarrays and as fluorescence donorsor acceptors in hybridization-based assays.

Example 77 Staining Cells with Tandem Dye-labeled Streptavidin

Jurkat cells are washed twice with 1% BSA/PBS and resuspended at aconcentration of 1×10⁷ cells/mL. The Jurkat cells are then incubated onice for 60 minutes with mouse anti human CD4 biotin (BiosourceInternational) at the recommended concentration of 10 μL for 1×10⁶cells. After incubation with the primary antibody, the cells are washedwith 1% BSA/PBS and incubated on ice for 30 minutes with 1 μg of eitherthe fluorescent streptavidin-phycoerythrin conjugate of Example 58, or astreptavidin conjugate of GIBCO'S RED 670. The cells are washed with 1%BSA/PBS, centrifuged, and resuspended with 400 μL of 1% BSA/PBS. Thesamples are analyzed on a FacsVantage flow cytometer exciting with the488-nm line of an argon laser, collecting the emission by a 700-nm longpass filter (XF-48). Using a FSC versus SSC dot plot the live cells aregated and the geometric mean of the fluorescence for FL3 is measured.The data is analyzed for both fluorescence and signal/noise ratio.

1. A method of analyzing a biological sample, wherein the methodcomprises combining a dye solution comprising a conjugate of abiological substance prepared from dye I,

wherein C is a non-conjugated chain of 10 to 50 linear atoms selectedfrom a group consisting of carbon and nitrogen that are furthersubstituted by a hydrogen, an alkyl having 1-20 carbons, a hydroxyl, ora carbonyl; X is CR₁₅R₁₆; n is 0 or 3; R₁, R₂ and R₃ are independently ahydrogen having 1-20 carbons, a cycloalkyl having 3-20 carbons, an aryl,a heteroaryl, an amino, an alkylamino, an arylamino, a thiol, analkylthio or a RGM; R₄-R₁₁ and R₁₃-R₁₆ are independently a hydrogen, analkyl having 1-20 carbons, a hydroxyl, an alkoxy having 1-20 carbons, afluorinated alkyl, a halogen, an alkylthiol, a sulfonyl, a carbonyl, ahydroxyl, an amino, an alkylthiol, a thiol, a sulfate, a phosphonate ora RGM; RGM is a carboxylic acid, an activated ester of a carboxylicacid, an acyl azide, an acyl nitrite, an imido ester, an isocyanate, anisothiocyanate, or a maleimide; wherein the biological substance isselected from a group consisting of a peptide, protein, nucleotide,nucleic acid, carbohydrate, lipid, membrane or cell, and wherein theconjugate is analyzed by detecting optical response upon use of anillumination source.
 2. The method according to claim 1, wherein RGM isan activated ester of a carboxylic acid, an imido ester, or a maleimide.3. The method according to claim 2, wherein either R₁₅ or R₁₆ is an RGM.4. The method according to claim 3, wherein R₄ is an RGM.
 5. The methodaccording to claim 4, wherein C is a non-conjugated chain of 10-50linear atoms selected from the group consisting of carbon and nitrogenthat are further substituted by a hydrogen and a carbonyl.
 6. The methodaccording to claim 5, wherein R₆-R₁₁ and R₁₃-R₁₄ are independently ahydrogen, a sulfate or a phosphonate.
 7. The method according to claim6, wherein the biological substance is a peptide or a protein.
 8. Themethod according to claim 6, wherein the biological substance is anucleotide or a nucleic acid.
 9. The method according to claim 6,wherein the biological substance is a carbohydrate or a lipid.
 10. Themethod according to claim 6, wherein the biological substance is amembrane or a cell.